Surface modified nanoparticles, methods of their preparation, and uses thereof for gene and drug delivery

ABSTRACT

A composition comprises a surface modified nanoparticle comprising a core comprising a material selected from the group consisting of organic materials, organometallic materials, inorganic materials, metals, metal oxides, and combinations thereof; and a surface branch covalently linked to the core having the general formula (3):

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of, and claims the benefit of,nonprovisional U.S. application Ser. No. 12/695991 entitled “SURFACEMODIFIED NANOPARTICLES, METHODS OF THEIR PREPARATION, AND USES THEREOFFOR GENE AND DRUG DELIVERY” filed on Jan. 28, 2010, issued as U.S. Pat.No. 8,226,985 on Jul. 24, 2012, herein incorporated by reference in itsentirety.

BACKGROUND

The present invention relates to surface modified nanoparticles, methodsof their preparation, and uses thereof for gene and drug delivery, andmore specifically, to nanoparticles comprising surface polymers derivedby a ring opening reaction.

The emerging field of nanomedicine offers opportunities in health careincluding curing disease and repairing damaged tissues. Polymerspossessing controlled functionalities, molecular weights,polydispersities, molecular architectures and topologies are expected toplay essential roles in the transport and delivery of drugs, genes, andother biologically active materials. Synthetic designs are sought thatprovide specific recognition and targeting capabilities of thecarrier-cargo complex. Ultimately, compartmentalized (e.g., layered)nanostructures are desired in which each compartment provides somefeature necessary for transport, delivery and/or release of a bio-activecargo.

Numerous polymer carriers have been prepared that are capable ofdelivering biologically active cargo both in vitro and in vivo,including both dynamic and static assemblies. Dynamic delivery vehiclesinclude micelles, vesicles, liposomes, etc. which self assemble insolution around the drug cargo and are subsequently delivered to thetarget. These nanostructures are in continuous dynamic equilibrium withthe monomeric cargo components. The micelles are constantly dissociatingand reassembling, which can be advantageous for cargo release, butlimiting to the circulatory lifetime of the complex. A dendrimer is anexample of a static, covalently bonded, core-shell system that islimited in cargo capacity and expensive to manufacture. Star-shapedmacromolecules circumvent some of these limitations, but star-shapedmacromolecular compositions (and synthetic approaches thereto) found inthe art offer limited versatility and utility with respect to thetransport, delivery and/or release of a bio-active cargo.

A continuing need exists for expanding the scope of macromolecularcompositions for gene and drug delivery, and methods of theirpreparation that provide synthetic design flexibility for tuning carrierproperties.

SUMMARY

According to one embodiment of the present invention, a compositioncomprises a surface modified nanoparticle comprising:

a core comprising a material selected from the group consisting oforganic materials, organometallic materials, inorganic materials,metals, metal oxides, and combinations thereof; and

a surface branch covalently linked to the core having the generalformula (3):

wherein

X^(d) is selected from the group consisting of —O—,

—S—, and combinations thereof, wherein R⁴ is a monovalent radicalcomprising 1 to 30 carbons,

j is an integer greater than or equal to 1,

each of X^(e), X^(f), and X^(g) is independently selected from the groupconsisting of —O—,

and —S—, wherein R⁶ is selected from the group consisting of hydrogenand monovalent hydrocarbon radicals comprising 1 to 30 carbons,

P′ comprises a first polymer comprising a backbone selected from thegroup consisting of polycarbonates, polyesters, polyureas,polycarbamates, polythiocarbamates, polythioureas, and combinationsthereof,

each R⁵ is independently selected from the group consisting of hydrogen,and monovalent hydrocarbon radicals comprising 1 to 30 carbons, and

each F′ is independently a monovalent radical.

In another embodiment, a method comprises:

independently covalently attaching by a ring opening reaction a firstcyclic carbonyl monomer independently to three or more nucleophilicsurface groups of a nanostructure, thereby forming a first modifiednanoparticle, the first modified nanoparticle comprising three or moreinitiator groups produced by the ring opening of the first cycliccarbonyl monomer; and

independently initiating a ring opening polymerization of a mixturecomprising one or more cyclic carbonyl monomers by the three or moreinitiator groups, thereby forming a surface modified nanoparticlecomprising three or more independent surface branches, wherein each ofthe three or more independent surface branches comprises a first polymerproduced by the ring opening polymerization;

wherein the first cyclic carbonyl monomer has the general formula (12):

wherein

each Y is independently selected from the group consisting of —O—, —NH—,

and —S—,

R⁵ and R⁶ are independent monovalent radicals selected from the groupconsisting of hydrogen and monovalent hydrocarbon radicals comprising 1to 30 carbons, and

M¹ is is a monovalent radical selected from the group consisting of—OR¹, —NHR¹, —NR¹R¹, and —SR¹, wherein R¹ is a monovalent radical.

In another embodiment, a method of preparing a loaded nanoparticle,comprises contacting a first aqueous mixture comprising the abovedescribed composition with a second aqueous mixture comprising abiologically active material.

In another embodiment, a method comprises:

independently attaching by a ring opening reaction a first cycliccarbonyl monomer to three or more nucleophilic surface groups of ananostructure, thereby forming a first modified nanoparticle, the firstmodified nanoparticle comprising three or more functional groups F′ andthree or more initiator groups; and

independently initiating by each of the three or more initiator groups aring opening polymerization of one or more cyclic carbonyl monomers,thereby forming a surface modified nanoparticle comprising three or moreindependent surface branches, wherein each of the three or moreindependent surface branches has the general formula (3):

wherein

X^(d) is a divalent radical selected from the group consisting of —O—,

—S—, and combinations thereof, R⁴ is a monovalent radical comprising 1to 30 carbons,

j is an integer greater than or equal to 1,

P′ is comprises a first polymer comprising a backbone selected from thegroup consisting of polycarbonates, polyesters, polyureas,polycarbamates, polythiocarbamates, polythioureas, and combinationsthereof,

each X^(e), X^(f), and X^(g) is independently selected from the groupconsisting of —O—,

—S—, and combinations thereof, wherein R⁶ is hydrogen or a monovalenthydrocarbon radical comprising 1 to 30 carbons,

each R⁵ is an independent monovalent radical selected from the groupconsisting of hydrogen and monovalent hydrocarbon radicals comprising 1to 30 carbons, and

each functional group F′ is independently a monovalent radical.

DETAILED DESCRIPTION

Efficient synthetic processes are disclosed for transforming a soft orhard pre-formed nanostructure, comprising a minimum of threenucleophilic surface groups, into a surface modified nanoparticlecomprising a tunable functional layer for gene and drug delivery. Atleast three nucleophilic surface groups of the surface modifiednanoparticle are each covalently linked to an independent surface branchcomprising a polymer chain derived by ring opening polymerization (ROP)of one or more cyclic carbonyl monomers, referred to herein as a ROPpolymer. The ROP polymer can comprise a homopolymer, random copolymer,block copolymer, or combination thereof, and can display amphiphilicproperties suitable for loading and carrying a biologically activematerial, such as a gene or a drug. The nanostructure can comprise avariety of materials, including but not limited to the group consistingof organic materials, organometallic materials, inorganic materials,metals, metal oxides, and combinations thereof. Nanostructures can alsocomprise clays and organoclays. The three or more nucleophilic surfacegroups are each capable of ring opening a first cyclic carbonyl monomer,and more particularly a first cyclic carbonate monomer. Exemplary cycliccarbonyl monomers include cyclic esters, cyclic carbonates, and cycliclactide monomers. Other cyclic carbonyl monomers include cyclic ureas,cyclic carbamates, cyclic thiocarbamates, and cyclic thioureas. Asurface branch comprising a ROP polymer is grown in one or more stepsfrom an initiator group formed by a ring opening reaction of the firstcyclic carbonyl monomer by a nucleophilic surface group of thenanostructure. The surface branches can be designed to comprise a broadrange of pendant functional groups, resulting in “functional layering”of the surface. The functional layers can include, for example,molecular tags/probes, reactive groups for covalently attaching abiologically active cargo (referred to as cargo), groups for releasing acargo, and/or linear polymers useful in forming reversible complexeswith a cargo. Compositions comprising the surface modified nanoparticlesare also disclosed.

In particular, each surface branch of the surface modified nanoparticleindependently comprises a peripheral first polymer and a linking groupthat covalently links the peripheral first polymer to a nucleophilicsurface group of the nanostructure. The linking groups have awell-defined functionality, and form one of the tunable functionallayers of the surface modified nanoparticles, in addition to the core ofthe nanostructure and the peripheral first polymer. The selection of anappropriate functionalized first cyclic carbonyl monomer for the linkinggroup, and one or more cyclic carbonyl monomers for the peripheral firstpolymer, which can include the first cyclic carbonyl monomer, allows oneto specifically tune the hydrophilic and/or hydrophobic balance, cargobinding properties, and release properties of the surface modifiednanoparticles for a given cargo and/or optimization of membranetargeting properties.

The nanostructure is represented by the general formula (1):

wherein the wavy line represents the core of the nanostructure, and eachX^(a) is independently covalently linked to the core. The nanostructurecomprises w′ nucleophilic surface groups X^(a), wherein w′ is greaterthan or equal to 3. Each X^(a) is independently selected from the groupconsisting of —OH, —NH₂, —NHR⁴, —SH, and combinations thereof, whereinR⁴ is a monovalent radical comprising 1 to 30 carbons. The nanostructurecan have a particle size of about 0.1 nm to about 500 nm, 0.1 to 200 nm,0.1 nm to 100 nm, 0.1 to 50 nm, 0.1 nm to 20 nm, and more particularly,0.1 to 10 nm.

The surface modified nanoparticle has three or more surface branches. Asurface branch has the general formula (2):

where it is understood that the dash on the left of X^(d) indicates thepoint of attachment to core of the nanostructure. Each X^(d) is aresidue independently derived from one of the three or more nucleophilicgroups X^(a). Each X^(d) is independently bound to the core (not shown)and a linking group,

derived from the ring opening of a first cyclic carbonyl monomer. X^(d)is a divalent radical selected from the group consisting of —O—,

—S—, and combinations thereof, wherein the dashes indicate the point ofattachment, and each R⁴ is independently a monovalent radical comprising1 to 30 carbons; j is an integer greater than or equal to 1; P′ is aperipheral first polymer, also referred to as first polymer, comprisinga polymer chain fragment derived by ring opening polymerization of oneor more cyclic carbonyl monomers. K′ further comprises a functionalgroup F′. When j is 1, from 3 to 20 bonds directly link the carbonylgroup and the first polymer P′. When j is greater than 1, each K′ can bederived by a ring opening reaction of the first cyclic carbonyl monomer,or a different cyclic carbonyl monomer. In an embodiment, j is 1.

More particularly, a surface branch has the general formula (3):

wherein X^(d), j, and P′ are defined as above; each X^(e), X^(f), andX^(g) are independently selected from the group consisting of —O—,

—S—, and combinations thereof, wherein the dashes indicate the point ofattachment, and R⁶ is a monovalent hydrocarbon radical comprising 1 to30 carbons; each R⁵ is independently a hydrogen or a monovalenthydrocarbon radical comprising 1 to 30 carbons; and each functionalgroup F′ is independently a monovalent radical. Each functional group F′can independently comprise a non-polymeric group or a polymeric group,herein referred to as an optional second polymer. The ring openingpolymerization to form the polymer chain fragment (ROP polymer chain) ofP′ is initiated by an initiator group formed by the ring opening of thefirst cyclic carbonyl monomer. More particularly, P′ comprises a firstpolymer comprising a backbone type selected from the group consisting ofpolycarbonates, polyesters, polyureas, polycarbamates,polythiocarbamates, polythioureas, and combinations thereof. The one ormore cyclic carbonyl monomers can include the first cyclic carbonylmonomer, if desired. The optional second polymer can also comprise asecond ROP polymer chain. P′ can further comprise a substituent groupselected from the group consisting of urea groups, carboxylic acidgroups, carboxylic acid salts, latent carboxylic acid groups, quaternaryamine groups, tertiary amine groups, poly(alkylene ether) groups, andcombinations thereof. When j is greater than 1, each R⁵ and each F′ canindependently comprise the same or different groups. More particularly,each F′ can independently comprise a substituent group selected from thegroup consisting of urea groups, carboxylic acid groups, carboxylic acidsalts, latent carboxylic acid groups, quaternary amine groups, tertiaryamine groups, poly(alkylene ether) groups, and combinations thereof. Ina preferred embodiment, j is 1, and each X^(e) and X^(f) is oxygen. Inan embodiment, F′ comprises a second polymer. In another embodiment, F′is hydrogen. In still another embodiment, F′ comprises a poly(alkyleneether) chain, and P′ comprises a polymer backbone selected from thegroup consisting of polyester, polycarbonate, and combinations thereof.

The first polymer and the optional second polymer can independentlycomprise a homopolymer, random copolymer, block copolymer, or acombination thereof. The first polymer and/or the optional secondpolymer can further independently comprise a chain fragment derived byradical polymerization (e.g., poly(meth)acrylates,poly(meth)acrylamides, polystyrenes), condensation polymerization (e.g.,polyamides, polyesters, polyacetals), transesterification polymerization(e.g., polyesters, polycarbonates), ring opening polymerization (e.g.,polyethers, polyesters, polycarbonates, polycarbamate,polythiocarbamate, polyurea, and polythiourea), or combinations thereof.The foregoing backbone types are intended to be exemplary and notlimiting. As shown further below in Schemes 1 and 2, the first polymerand the second polymer can be attached to the nanostructure in reverseorder; that is, the ring opening polymerization to form the firstpolymer can be initiated prior to or after covalent attachment of thesecond polymer to the nanostructure.

The first polymer and/or the optional second polymer can independentlycomprise an optional end cap group (ECG). In an embodiment, the optionalend cap group comprises a moiety selected from the group consisting ofalkyl ester groups, aryl ester groups, poly(alkylene ether) groups,thiol groups, amine groups, carboxylic acid groups, quaternary aminegroups, functional moieties capable of targeting specific cell types,such as galactose and mannose, and combinations thereof.

A divergent method of forming a surface modified nanoparticle isillustrated by the non-limiting example of Scheme 1, wherein thenanostructure is treated initially with a first cyclic carbonyl monomerthat does not comprise a pendant polymer group (i.e., Fg₁ does notcomprise a polymer).

Accordingly, a nucleophilic surface group is initially reacted with afirst cyclic carbonyl monomer in a ring opening reaction to form a firstmodified nanoparticle, which comprises an initiator group for anotherring opening reaction, or a ROP polymerization of one or more cycliccarbonyl monomers, which can include the first cyclic carbonyl monomer.In the example shown, the nanostructure has w′ nucleophilic surfacegroups, where w′≧3. In this example, a second cyclic carbonyl monomerand a third cyclic carbonyl monomer are ring opened sequentially,thereby forming a block copolymer chain, as shown in the second modifiednanoparticle and third modified nanoparticle, respectively. K′ and P′ inScheme 1 of the third modified nanoparticle correspond to K′ and P′,respectively, in general formula (2). Alternatively, the second cycliccarbonyl monomer and a third cyclic carbonyl monomer can be combined asa mixture to form a random copolymer chain by ring openingpolymerization (not shown). The functional layering arises from the corecomposition of the nanostructure and the different side chain functionalgroups Fg₁, Fg₂, Fg₃ . . . etc., that can be added to the growingsurface branch. Functional groups Fg₁, Fg₂, Fg₃ . . . etc. can furtherbe modified after the ring opening reaction. In an embodiment, one ormore of the one or more cyclic carbonyl monomers other than the firstcyclic carbonyl monomer comprises a poly(alkylene ether) side chaingroup.

As shown in the example of Scheme 1, the ring opening reaction of thefirst cyclic carbonyl monomer can be performed with or without acatalyst. In the absence of the catalyst, the primary amine group of thenanostructure reacts by ring opening the first cyclic carbonyl monomerwithout inducing polymerization (i.e., x/w′=1 in the first modifiednanoparticle). When a catalyst is present, the primary amine group ofthe nanostructure can initiate ring opening polymerization the firstcyclic carbonyl monomer (i.e., x is a positive integer multiple of n).In an embodiment, the first ring opening reaction is performed without acatalyst, and without polymerizing the first cyclic carbonyl monomer. Ineither scenario, the ring opening reaction forms a fragment comprisingan initiator group, which can be used for ring opening polymerization(ROP initiator group), and a pendant side chain functional group Fg₁.The initiator group in the above example is a hydroxyl group. Eachadditional ring opening reaction potentially adds a new ROP initiatorgroup to the peripheral end of the growing polymer chain. If desired,the catalyst can be excluded in one or more of the subsequent ringopening reaction steps to add a single ring opened cyclic carbonylmonomer unit at the peripheral end of the growing chain. In this manner,the surface branch can be tailored for many properties, includingbinding strength to a biologically active material, molecular weight,hydrophilic/hydrophobic balance, membrane recognition, solutionstability, biodegradability, and particle size. No limitation is placedon the number of cyclic carbonyl monomers, the number of ring openingsteps, the backbone type, the number of polymer blocks, or the sidechain functionality, providing that the modified nanoparticle has thedesirable carrier properties, which can include cytotoxicity, aqueousdispersion properties, hemolysis properties, and cargo releaseproperties. In an embodiment, the surface branch comprises a firstpolymer comprising a pendant functional group selected from the groupconsisting of urea groups, carboxylic acid groups, carboxylic acidsalts, latent carboxylic acid groups, quaternary amine groups, tertiaryamine groups, and combinations thereof. Other possible functional groupsare listed in the cyclic carbonyl monomers of Table 1.

For simplicity, all examples herein assume the ideal case that all w′ ofthe nucleophilic surface groups react and, therefore, the length ofpolymeric blocks may be described by the division of the number of molesof monomer units (e.g., x, y, z . . . etc.) by the number ofnucleophilic surface groups (w′). However, the reaction of 100% of thenucleophilic surface groups is not a requirement for successfulimplementation of the invention. The number of reacted nucleophilicsurface groups can be greater than or equal to three and less than orequal to w′ when w′ is greater than three. In some instances, from morethan 0% to less than 100% of the nucleophilic surface groups undergoring opening reaction with the first cyclic carbonyl monomer. In theseinstances, the actual length of polymeric blocks can be longer than thatcalculated for the ideal case (e.g., x/w′). Unreacted nucleophilicsurface groups can serve as additional reaction or initiator sitesduring subsequent synthetic processes. Therefore, it is advantageousthat a high percentage of the nucleophilic surface groups undergo thering opening reaction with the first cyclic carbonyl monomer.

In an embodiment, each ring opening reaction after the first ringopening reaction occurs in the presence of a catalyst, particularly anorganic catalyst, resulting in ring opening polymerization. If performedsequentially, the ROP polymer chain comprises a block copolymer. In anembodiment, the ROP block copolymer comprises a polycarbonate backbone.In another embodiment, the ROP block copolymer comprises a polyesterbackbone. In another embodiment the ROP block copolymer comprises abackbone selected from the group consisting of polycarbonate, polyester,and combinations thereof.

Also shown in Scheme 1 is a polymer chain (third modified nanoparticle),end capped with an acetyl group using acetic anhydride. The optional endcap group can impart stability and useful functionality to the finalstructure. For example, a more complex end cap group comprising agalactose moiety can potentially be useful in targeting liver cells. Endcapping agents are numerous, and methods of their use are wellestablished in the polymer art. End capping agents can be selected basedon the functionality desired and their intended use. In an embodiment,the surface modified nanoparticle comprises a surface branch wherein theperipheral end unit comprises a quaternary amine. In another embodiment,the peripheral end unit comprises a poly(alkylene ether) chain.

A convergent method of forming a surface modified nanoparticle isillustrated by the non-limiting example of Scheme 2, wherein thenanostructure is treated initially with a first cyclic carbonyl monomercomprising a pendant polymer group (second polymer).

In the above example, mono-end capped poly(ethylene glycol) (MPEG) isderivatized on the free hydroxyl end by reaction with a cyclic carbonylcompound, MTCX, wherein X is a suitable leaving group, thereby formingthe first cyclic carbonyl monomer MTCOMPEG comprising a pendant MPEGgroup (second polymer). A nanostructure comprising w′ nucleophilicsurface groups, where w′≧3, undergoes a ring opening reaction withMTCOMPEG, thereby forming a first modified nanoparticle. The firstmodified nanoparticle comprises an initiator group, in this example ahydroxyl group, capable of initiating ring opening polymerization of asecond cyclic carbonyl monomer. In this example, the second cycliccarbonyl monomer is L-lactide (LLA, stereochemistry not shown), and ringopening polymerization of LLA produces a second modified nanoparticlecomprising a poly(L-lactide) chain fragment (first polymer). Thepoly(L-lactide) chain fragment comprises a living end unit (a hydroxylgroup) capable of initiating ring opening polymerization of a thirdcyclic carbonyl monomer, if desired. K′ and P′ in Scheme 2 correspond toK′ and P′, respectively, of general formula (2). As shown in thisexample, the surface branch of the surface modified nanoparticlecomprises a hydrophobic poly(L-lactide) polymer fragment (first polymer)and a hydrophilic MPEG polymer fragment (second polymer) that are linkedto the nanostructure through the fragment derived from MTCX. The aboveexample is not meant to be restrictive. As stated above, the secondpolymer can comprise a wide variety of polymer types. In an embodiment,the second polymer comprises a poly(alkylene ether) chain, as shown inScheme 2. In another embodiment, one or more cyclic carbonyl monomersused in preparing the first polymer comprises a pendant poly(alkyleneether) chain. In another embodiment, the surface branch formed by theconvergent method comprises a functional group selected from the groupconsisting of urea groups, carboxylic acid groups, carboxylic acidsalts, latent carboxylic acid groups, quaternary amine groups, tertiaryamine groups, and combinations thereof.

The first polymer and/or the second polymer can further comprise an endcap group (e.g., the methoxy group of MPEG (shown)), a living end unit,or a protected end unit that can be deprotected after the ring openingreaction with the nanostructure. The deprotected end unit can comprise,for example, an initiator group for ring opening polymerization of acyclic carbonyl monomer. Alternatively, the deprotected end unit can bemodified to introduce a biologically useful group to the end unit, suchas a galactose moiety for liver cell recognition. In an embodiment, thefirst polymer and/or the second polymer is end capped with apoly(alkeylene ether) chain.

For simplicity, all examples herein assume the ideal case that all w′ ofthe nucleophilic surface groups react and, therefore, the length ofpolymeric blocks may be described by the division of the number of molesof monomer units (e.g., x, y, z . . . etc.) by the number ofnucleophilic surface groups (w′). However, the reaction of 100% or thenucleophilic surface groups is not a requirement for successfulimplementation of the invention. In the case of the convergent approach,only some of the nucleophilic surface groups may react due to sterichindrance by the pendant polymer group attached to the cyclic carbonylgroup. In these cases, the actual length of polymeric blocks wouldlonger that that calculated for the ideal case (e.g., x/w′). When w′ isgreater than three, the number of reacted nucleophilic surface groupscan be greater than or equal to three and less than or equal to w′.Unreacted nucleophilic surface groups can serve as additional reactionor initiator sites during subsequent synthetic processes. Therefore, itis advantageous if the degree of reaction of the nucleophilic surfacegroups is high.

Pre-Formed Nanostructures.

The nanostructure can be selected from a wide variety of materials, withthe proviso that the nanostructure has a minimum of three nucleophilicsurface groups, and the surface modified nanoparticle has the desiredcarrier properties, cytotoxicity, biodegradability, hemolyticproperties, and/or release properties for a given biologically activecargo. The bulk phase of the nanostructure can comprise, as non-limitingexamples, organic material, organometallic material, inorganic material,metal, metal oxide, clay, organoclay, or combinations of the foregoing.A particular inorganic material can be selected from the groupconsisting of cobalt, iron, and combinations thereof. The organicmaterial can comprise, for example, crosslinked polymers, latexes,hyperbranched polymers, dendrimers, dendrons, cyclodextrins, materialspossessing lower critical solution temperature (LCST) transitions inwater at from about 0° C. to about 50° C., or combinations of theforegoing materials. The nanostructure can comprise between 3 and 64(inclusive) or more nucleophilic surface groups. Exemplary nucleophilicsurface groups include primary alcohols, secondary alcohols, primaryamines, secondary amines, primary thiols, secondary thiols, orcombinations of the foregoing functional groups.

In an embodiment, the nanostructure comprises a dendritic polymermaterial comprising 3 or more nucleophilic surface groups. Dendrimersare monodisperse, tree-like or generational polymer structures preparedin a stepwise process. Dendrimers are constructed one monomer layer, or“generation,” at a time. Each dendrimer comprises a multifunctional coremolecule, and each functional site of the core molecule has a dendriticwedge attached thereto. The functional sites of the core molecule canbe, for example, primary amine groups or another reactive group fromwhich a dendritic wedge can be grown using a step-wise syntheticprocess. The core molecule is referred to as “generation 0.” Eachsuccessive repeat unit along all branches forms the next generation,“generation 1,” “generation 2,” and so on until the nth terminatinggeneration. The terminating generation comprises the nucleophilicsurface groups used herein to grow a polymer from the dendrimer surfacegroups, or attach a pre-formed polymer to the dendrimer surface groups.More particularly, the dendrimer can comprise between 3 and 64(inclusive) or more nucleophilic surface groups capable of ring openinga first cyclic carbonyl monomer. In a particular embodiment, the firstcyclic carbonyl monomer is a cyclic carbonate monomer.

The core molecule from which the dendrimer is derived has the generalformula (4):R′—(X′)_(n″)  (4),wherein R′ is group comprising from 1 to 20 carbons; each X′ is anucleophilic group independently selected from the group consisting of—OH, —NH₂, —NR⁷, and —SH, and combinations thereof, wherein R⁷ is amonovalent radical comprising 1 to 30 carbons; and n″ is an integer from2 to 10. R′ can comprise a moiety selected from the group consisting ofhydrocarbon chains, aliphatic rings, heterocyclic rings, aromatic rings,and combinations thereof. R′ can further comprise a functional groupselected from the group consisting of ether, amide, ester, secondaryamine, tertiary amine, quaternary amine, sulfide, disulfide, sulfonate,urea, carbamate, carbonate, phosphate, phosphonate, and combinationsthereof. In an embodiment, the core molecule is a diaminoalkanecomprising 2 to 20 carbons.

The first generational growth step adds a minimum of two branches toeach X′ group of the core molecule. As a non-limiting example, if thecore molecule is 1,4-diaminobutane (DAB), each X′ is —NH₂. The firstgeneration growth step can be the attachment of two aminopropyl groupsto each X′ group, as follows:

DAB4 is a commercially available first generational polypropyleniminedendrimer, sold by Sigma Aldrich. In a second generation growth step,two aminopropyl chains are attached to each of the four primary aminogroups of DAB4, adding four additional branches to the structure,producing DAB8. This process can be repeated for each growth step,doubling the terminal branches in each generation, resulting in amonodisperse structure. Thus, DAB 8, DAB16, DAB32, and DAB64, are thesecond, third, fourth and fifth generation dendrimers derived from DAB,and have 8, 16, 32 and 64 terminal primary amine groups, respectively.

DAB4 is for illustrative purposes and is not meant to be limiting. Eachbranch of the dendrimer can independently comprise any of the moietiesand/or functional groups defined above for R′ of formula (4). Moreparticularly, the dendrimer can comprise branches comprising heteroatomsselected from nitrogen, oxygen, sulfur, phosphorous, and combinationsthereof. The outermost branches comprise end groups selected from thegroup consisting of primary amines, secondary amines, primary alcohols,secondary alcohols, primary thiols, secondary thiols, and combinationsof the foregoing, that can react with a cyclic carbonyl monomer by aring opening reaction.

Other commercially available dendrimers are based on monodispersepolyamidoamines (PAMAM). Surface groups include amidoethanol(—CONH(CH₂)₂OH), amidoethylethanolamine (—CONH(CH₂)₂NH(CH₂)₂OH),amidoamine (—CONH(CH₂)₂NH₂), tris(hydroxymethyl)amidomethane(—CONHCH₂(CH₂OH)₃), bi-functional surface groups comprising amine andhydroxyl groups, and carboxylic acid surface groups. Core molecules usedto prepare PAMAM dendrimers include 1,2-ethylenediamine,1,4-diaminobutane, 1,6-diaminohexane, 1,12-diaminododecane, andcystamine.

Nanostructures can also include polydisperse hyperbranchedmacromolecules such as those based on 2,2-bis(methylol)propionic acid(bisMPA) chemistry, also commercially available from Sigma Aldrich.These generally have branches comprising polyethers, polyesters, andcombinations thereof, and terminal branches comprising primary alcoholgroups.

Polyethers.

A polyether chain can provide an important means of introducinghydrophilicity into the surface modified nanoparticle. As stated above,a mono end capped polyether alcohol can be employed as an initiator forring opening polymerization of a cyclic carbonyl monomer, therebyintroducing a main chain hydrophilic block into the resulting ROPpolymer.

The polyether alcohol can be a poly(alkylene glycol) of the generalformula (5):HO—[C(R⁷)₂(C(R⁷)₂)_(a′)C(R⁷)₂O]_(n)—H  (5),Wherein a′ is 0 to 8, n is an integer from 2 to 10000, and each R⁷ isindependently a monovalent radical consisting of hydrogen and an alkylgroup of 1 to 30 carbons. Thus, the ether repeat unit comprises 2 to 10backbone carbons between each backbone oxygen. More particularly, thepoly(alkylene glycol) can be a mono endcapped poly(alkylene glycol),represented by the formula (6):R⁸O—[C(R⁷)₂(C(R⁷)₂)_(a′)C(R⁷)₂O]_(n)—H  (6),wherein R⁸ is a monovalent hydrocarbon radical comprising 1 to 20carbons.

As non-limiting examples, the polyether alcohol can be a poly(ethyleneglycol) (PEG), having the structure HO—[CH₂CH₂O]_(n)—H, wherein theether repeat unit CH₂CH₂O (shown in the brackets) comprises two backbonecarbons linked to a backbone oxygen. The polyether alcohol can also be apolypropylene glycol) (PPG) having the structure HO—[CH₂CH(CH₃)O]_(n)—H,where the ether repeat unit CH₂CH(CH₃)O comprises two backbone carbonslinked to a backbone oxygen with a methyl side-chain. An example of monoend capped PEG is the commercially available monomethyl end capped PEG,wherein R⁸ is a methyl group. Other examples include poly(oxetane),having the structure HO—[CH₂CH₂CH₂O]_(n)—H, and poly(tetrahydrofuran),having the structure HO—[CH₂(CH₂)₂CH₂O]_(n)—H

The mono end capped poly(alkylene glycol) can comprise more elaboratechemical structures, represented by the general formula (7):Z′—[C(R⁷)₂(C(R⁷)₂)_(a′)C(R⁷)₂O]_(n-1)—H  (7),wherein Z′ is a monovalent radical including the backbone carbons andoxygen of the end repeat unit, and can have 2 to 100 carbons. Thefollowing non-limiting examples illustrate mono end-derivatization ofpoly(ethylene glycol) (PEG). As described above, one end repeat unit ofPEG can be capped with a monovalent hydrocarbon group having 1 to 20carbons, such as the monomethyl PEG (MPEG), wherein Z′ is MeOCH₂CH₂O— asshown further above for MPEG in Scheme 2. The dash on the end of theMeOCH₂CH₂O— indicates the point of attachment to the polyether chain. Inanother example, Z′ includes a thiol group, such as HSCH₂CH₂O—, or athioether group, such as MeSCH₂CH₂O—. In another example, one end unitof PEG is an aldyhyde, wherein Z′ can be OCHCH₂CH₂O—. Treating thealdehyde with a primary amine produces an imine, wherein Z′ isR⁹N═CHCH₂CH₂O—. R⁹ is a monovalent radical selected from hydrogen, analkyl group of 1 to 30 carbons, or an aryl group comprising 6 to 100carbons. Continuing, the imine can be reduced to an amine, wherein Z′ isR⁹NHCH₂CH₂CH₂O—. In another example, one end repeat unit of PEG can beoxidized to a carboxylic acid, wherein Z′ is HOOCCH₂O—. Using knownmethods the carboxylic acid can be converted to an ester, wherein Z′becomes R⁹OOCCH₂O—. Alternatively, the carboxylic acid can be convertedto an amide, wherein Z′ becomes R⁹NHOCCH₂O—. Many other derivatives arepossible. In a particular embodiment, Z′ is a group comprising abiologically active moiety that interacts with a specific cell type. Forexample, the Z′ group can comprise a galactose moiety which specificallyrecognizes liver cells. In this instance, Z′ has the structure:

where L′ is a divalent linking group comprising 2 to 50 carbonscontaining the end repeat unit. The hyphen on the right side of L′indicates the attachment point to the polyether chain. Z′ can compriseother biologically active moieties such as mannose.

A polyether alcohol employed as an initiator for a ring openingpolymerization can comprise a poly(alkylene glycol) or amono-derivatized poly(alkylene glycol). The polyether alcohol initiatorcan comprise a mono-derivatized end repeat unit; alternatively, themono-derivatized end repeat unit can be formed after the ring openingpolymerization.

The number average molecular weight of the polyether alcohol can be from100 to 100,000, more specifically 100 to 10000, and even morespecifically, 100 to 5000.

Cyclic Carbonyl Monomers.

The surface branch of the surface modified nanoparticle comprises apolymer chain fragment that in whole or in part is derived by ringopening polymerization of one or more cyclic carbonyl monomers.

A cyclic carbonyl monomer can have the general formula (8):

wherein t is an integer from 0 to 6, and when t is 0 carbons labeled 4and 6 are linked together by a single bond. Each Y is a divalent radicalindependently selected from the group consisting of —O—, —S—,

wherein the dashes “—” indicate the point of attachment in the ring. Thelatter group is also expressed herein as —N(Q¹)- and —C(Q¹)₂-. Each Q¹is an independent monovalent radical. Each Q¹ group can independently bebranched or non-branched. As examples, each Q¹ group can independentlybe selected from the group consisting of hydrogen, alkyl groupscomprising 1 to 100 carbons, and aryl groups comprising 6 to 100carbons. In an embodiment, at least one Q¹ group is a group other thanhydrogen. A Q¹ group can have the structure

wherein M¹ is a monovalent radical, polymeric or non-polymeric. Asexamples, each M¹ can independently be selected from the groupconsisting of —R¹, —OR¹, —NHR¹, —NR¹R¹, and —SR¹ wherein the dashrepresents the point of attachment, and R¹ is an independent polymericor non-polymeric monovalent radical. As examples, each R¹ can beindependently selected from the group consisting of alkyl groupscomprising 1 to 100 carbons, and aryl groups comprising 6 to 100carbons. Each Q¹ group can further independently comprise one or moreadditional functional groups selected from the group consisting ofketone groups, aldehyde groups, alkene groups, alkyne groups,cycloaliphatic rings comprising 3 to 10 carbons, heterocylic ringscomprising 2 to 10 carbons, ether groups, amide groups, ester groups,carboxylic acid groups, urea groups, and combinations of the foregoingadditional functional groups. The heterocyclic ring can comprise oxygen,sulfur and/or nitrogen. Two or more Q¹ groups can together form a ring.In an embodiment, one or more of the Q¹ groups comprises a monovalenturea radical. In another embodiment, one or more of the Q¹ groupscomprise a latent carboxylic acid group capable of being converted to acarboxylic acid after ring-opening polymerization. In anotherembodiment, one or more of the Q¹ groups comprises a functional groupcapable of reacting with a tertiary amine to form a quaternary amine. Inan embodiment, each Q¹ is independently selected from the groupconsisting of hydrogen, alkyl groups comprising 1 to 100 carbons, andaryl groups comprising 6 to 100 carbons.

A cyclic carbonyl monomer can have the general formula (9):

wherein each Q² and Q³ is an independent monovalent radical and R² is amonovalent radical, polymeric or non-polymeric. As examples, each Q² andQ³ can be independently selected from the group consisting of hydrogen,halides, alkyl groups having 1 to 100 carbons, and aryl groups having 6to 100 carbons. When Q² and Q³ are not hydrogen, Q² and Q³ representpendant moieties to the cyclic carbonyl ring that become side chains tothe ROP polymer chain. The —CO₂R² group also becomes a side chain to theROP polymer after ring opening polymerization. In an embodiment, each Q²is hydrogen and Q³ is a methyl or ethyl group. In an embodiment, R²comprises a monovalent urea radical. In another embodiment, R² comprisesa latent carboxylic acid group capable of being converted to acarboxylic acid after ring-opening polymerization. In anotherembodiment, R² comprises a functional group capable of reacting with atertiary amine to form a quaternary amine. In yet another embodiment, R²comprises a second polymer.

A cyclic carbonyl monomer can have the general formula (10):

wherein each Q⁴ is an independent monovalent radical, and u is aninteger from 1 to 8. As examples, each Q⁴ can independently be selectedfrom the group consisting of hydrogen, halides, alkyl groups comprising1 to 100 carbons, aryl groups comprising 6 to 100 carbon atoms, andgroups having the structure

wherein M¹ is a monovalent radical, polymeric or non-polymeric. Asexamples, M¹ can be selected from the group consisting of —R¹, —OR¹,—NHR¹, —NR¹R¹, and —SR¹ wherein the dash represents the point ofattachment, and R¹ is a monovalent radical, polymeric or non-polymeric.As examples, each R¹ can be independently selected from the groupconsisting of alkyl groups comprising 1 to 100 carbons, and aryl groupscomprising 6 to 100 carbons. When Q⁴ is not hydrogen, Q⁴ represents apendant moiety to the cyclic carbonyl ring that becomes a side chain tothe ROP polymer after ring opening polymerization. The lactone ring canoptionally comprise a carbon-carbon double bond; that is, optionally, a

group of formula (10) can independently represent a

or a

group. The lactone ring can also comprise a heteroatom not linked to thering carbonyl or ring oxygen, such as oxygen, nitrogen, sulfur, or acombination thereof; that is, optionally a

group of formula (10) can independently represent a —O—, —S—, or —NR¹—group. In an embodiment, u is an integer from 1 to 6 and each Q⁴ ishydrogen. In an embodiment, one or more of the Q⁴ groups comprises amonovalent urea radical. In another embodiment, one or more of the Q⁴groups comprise a latent carboxylic acid group capable of beingconverted to a carboxylic acid after ring opening polymerization. Inanother embodiment, one or more of the Q⁴ groups comprises a functionalgroup capable of reacting with a tertiary amine to form a quaternaryamine.

The cyclic carbonyl monomer can have the general formula (11):

wherein each Q⁵ is an independent monovalent radical. As examples, eachQ⁵ can independently be selected from the group consisting of hydrogen,halides, alkyl groups comprising 1 to 100 carbons, aryl groupscomprising 6 to 100 carbon atoms, and groups having the structure

wherein M¹ is a monovalent radical, polymeric or non-polymeric, and eachv is independently an integer from 1 to 6. As examples, M¹ can beselected from the group consisting of —R¹, —OR¹, —NHR¹, —NR¹R¹, and —SR¹wherein the dash represents the point of attachment, and R¹ is amonovalent radical, polymeric or non-polymeric. As examples, each R¹ canbe independently selected from the group consisting of alkyl groupscomprising 1 to 100 carbons, and aryl groups comprising 6 to 100carbons. Each Q⁶ is an independent monovalent radical. As examples, eachQ⁶ can independently be selected from the group consisting of hydrogen,alkyl groups having 1 to 100 carbons, and aryl groups having 6 to 100carbons. When Q⁵ and Q⁶ are not hydrogen, Q⁵ and Q⁶ represent pendantmoieties to the cyclic carbonyl ring that become side chains to the ROPpolymer after ring opening polymerization. In an embodiment, each v is1, each Q⁵ is hydrogen, and each Q⁶ is a hydrocarbon group comprising 1to 6 carbons. In an embodiment, one or more of the Q⁵ and/or Q⁶ groupscomprises a monovalent urea radical. In another embodiment, one or moreof the Q⁵ and/or Q⁶ groups comprises a latent carboxylic acid groupcapable of being converted to a carboxylic acid after ring-openingpolymerization. In another embodiment, one or more of the Q⁵ and/or Q⁶groups comprises a functional group capable of reacting with a tertiaryamine to form a quaternary amine.

The first cyclic carbonyl monomer has the general formula (12):

wherein each Y is independently selected from the group consisting of—O—, —NH—,

and —S—, R⁵ and R⁶ are independent monovalent radicals selected from thegroup consisting of hydrogen and monovalent hydrocarbon radicalscomprising 1 to 30 carbons, and M¹ is selected from the group consistingof —OR¹, —NHR¹, —NR¹R¹, and —SR¹ wherein the dash represents the pointof attachment, and R¹ is a monovalent radical. M¹ can comprise anon-polymeric group or a second polymer.

Non-limiting examples of latent carboxylic acids include esters that canbe hydrolyzed under mild conditions (e.g., trifluoroethyl ester,pentafluorophenyl ester, or p-nitrophenyl ester, N-hydroxysuccinimimideester, trimethylsilyl ester, tetrahydropyranyl ester). Other latentcarboxylic acids include thermally labile tertiary esters (e.g., t-butylesters). Still other latent carboxylic acids include esters capable ofbeing reductively cleaved using hydrogen and a suitable catalyst (e.g.,benzyl esters, cleavable by H₂/Pd—C). In an embodiment, the latentcarboxylic acid group is any carboxylic ester that can be converted to acarboxylic acid by hydrogenation using a suitable catalyst. One exampleis the benzyl ester of MTCOBn.

The benzyl ester of MTCOBn can be cleaved to a carboxylic acid usingH₂/Pd—C after the ring opening polymerization. In another embodiment, alatent carboxylic acid excludes primary or secondary aliphatichydrocarbon chain esters which cannot be selectively cleaved in thepresence of the backbone ester and/or carbonate structures.

Another example of a latent carboxylic acid group is an acetal-protectedcarboxylic acid group, herein also referred to as an acetal ester group.The acetal ester group has the general formula (13):

wherein * represents the site of attachment to a cyclic carbonyl moiety,and R^(c) and R^(d) are monovalent radicals independently comprisingfrom 1 to 20 carbons. In an embodiment, R^(c) is methyl and R^(d) isethyl. An example of cyclic carbonyl compound comprising an acetal esteris MTCOEE:

When copolymerized into the polymer, repeat units derived from MTCOEEcomprise a side chain acetal ester that is readily deprotected in theacidic endosomal environment. Once released into the cytoplasm, theresulting carboxylic acid groups of the cationic polymer can bedeprotonated,

Additional cyclic carbonyl monomers of formulas (9), (10), and (11) arelisted in Table 1.

TABLE 1

The cyclic carbonyl monomers can be purified by recrystallization from asolvent such as ethyl acetate or by other known methods of purification,with particular attention being paid to removing as much water aspossible from the monomer. The monomer moisture content can be from 1 to10,000 ppm, 1 to 1,000 ppm, 1 to 500 ppm, and most specifically 1 to 100ppm, by weight of the monomer.

The cyclic carbonyl monomers can also comprise isotopically enrichedforms of the cyclic carbonyl monomers. These include functional groupscomprising elements selected from the group consisting of ¹³C, ¹⁴C, ¹⁵N,deuterium, tritium, and combinations thereof. The cyclic carbonylmonomers can also comprise a radioactive moiety suitable for targeting aspecific cell type, such as a cancer cell. The radioactive moiety cancomprise a heavy metal radioactive isotope.

The cyclic carbonyl monomer can comprise a reactive monovalent leavinggroup that when treated with a tertiary amine, produces a quaternaryamine. Reactive monovalent leaving groups include alkyl halides (e.g.,alkyl chlorides, alkyl bromides, or alkyl iodides), sulfonate esters(e.g., tosylates, or mesylates), epoxides, and oxetanes. Reaction withthe tertiary amine is generally performed after the ring openingreaction when the reactive monovalent leaving group occupies a sidechain position in the ROP polymer.

The tertiary amine can comprise a single nitrogen such as atrialkylamine, including but not limited to trimethylamine,triethylamine, tripropylamine, and the like. The tertiary amine canfurther comprise additional functional groups, in particular acarboxylic acid group, for example 3-(N,N-dimethylamino)propionic acid.In such instances, the cationic polymer will comprise first repeat unitscomprising a side chain moiety comprising a quaternary amine and acarboxylic acid group.

The tertiary amine can also comprise isotopically enriched versions ofthe tertiary amine, such as trimethylamine-¹⁴C, trimethylamine-¹⁵N,trimethylamine-¹⁵N, trimethyl-¹³C₃-amine, trimethyl-d₉-amine, andtrimethyl-d₉-amine-¹⁵N. The tertiary amine can also comprise aradioactive moiety suitable for targeting a specific cell type, such asa cancer cell. The radioactive moiety can comprise a heavy metalradioactive isotope.

The tertiary amine can be a bis-tertiary amine of the general formula(14):

where L″ is a divalent linking group comprising 2 to 30 carbons, andeach monovalent R_(b) group is independently selected from alkyl groupscomprising 1 to 30 carbons or aryl groups comprising 6 to 30 carbons.Each R^(b) group can independently be branched or non-branched. EachR^(b) group can independently comprise additional functional groups suchas a ketone group, aldehyde group, hydroxyl group, alkene group, alkynegroup, cycloaliphatic ring comprising 3 to 10 carbons, heterocylic ringcomprising 2 to 10 carbons, ether group, amide group, ester group, andcombinations of the foregoing additional functional groups. Theheterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two ormore R^(b) groups can also together form a ring. Representative L″groups include —(CH₂)_(z′)— where z′ is an integer from 2 to 30,—(CH₂CH₂O)_(z″)CH₂CH₂— where z″ is an integer from 1 to 10,—CH₂CH₂SCH₂CH₂—, —CH₂CH₂SSCH₂CH₂—, —CH₂CH₂SOCH₂CH₂—, and—CH₂CH₂SO₂CH₂CH₂—. L″ can further comprise a monovalent or divalentcycloaliphatic ring comprising 3 to 20 carbons, a monovalent or divalentaromatic ring comprising 6 to 20 carbons, a ketone group, aldehydegroup, hydroxyl group, alkene group, alkyne group, a heterocylic ringcomprising 2 to 10 carbons, ether group, amide group, ester group, andcombinations of the foregoing functional groups. The heterocyclic ringcan comprise oxygen, sulfur and/or nitrogen. The bis-tertiary amine canalso comprise isotopically enriched forms of the bis-tertiary amine,such as deuterium, carbon-13, and/or nitrogen-15 enriched forms thereof.

More specific bis-tertiary amines includeN,N,N′,N′-tetramethyl-1,2-ethanediamine (TMEDA),N,N,N′,N′-tetramethyl-1,3-propanediamine (TMPDA),N,N,N′,N′-tetramethyl-1,4-butanediamine (TMBDA),N,N,N′,N′-tetraethyl-1,2-ethanediamine (TEEDA),N,N,N′,N′-tetraethyl-1,3propanediamine (TEPDA),1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),N,N,N′,N′-tetraethyl-1,4-butanediamine (TEBDA), 4-dimethylaminopyridine(DMAP), 4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations thereof.In an embodiment, the bis-tertiary amine is TMEDA.

The above-described cyclic carbonyl monomers undergo ring-openingpolymerization to form a ROP polymers in atactic, syndiotactic orisotactic forms. The particular tacticity depends on the cyclicmonomer(s), isomeric purity, and the reaction conditions.

The reaction mixture for the ring opening polymerization comprises oneor more cyclic carbonyl monomers; a catalyst; an optional accelerator;an optional solvent, and an initiator. The ring opening polymerizationis generally conducted in a reactor under inert atmosphere such asnitrogen or argon. The polymerization can be performed by solutionpolymerization in an anhydrous non-protic solvent such as benzene,toluene, xylene, cyclohexane, n-hexane, dioxane, chloroform anddichloroethane, or by bulk polymerization. The reaction temperature canbe from about ambient temperature to 250° C. Generally, the reactionmixture is heated at atmospheric pressure for 0.5 to 72 hours to effectpolymerization, forming a second mixture.

Exemplary catalysts for the ring opening polymerization include metaloxides such as tetramethoxy zirconium, tetra-iso-propoxy zirconium,tetra-iso-butoxy zirconium, tetra-n-butoxy zirconium, tetra-t-butoxyzirconium, triethoxy aluminum, tri-n-propoxy aluminum, tri-iso-propoxyaluminum, tri-n-butoxy aluminum, tri-iso-butoxy aluminum, tri-sec-butoxyaluminum, mono-sec-butoxy-di-iso-propoxy aluminum, ethyl acetoacetatealuminum diisopropylate, aluminum tris(ethyl acetoacetate), tetraethoxytitanium, tetra-iso-propoxy titanium, tetra-n-propoxy titanium,tetra-n-butoxy titanium, tetra-sec-butoxy titanium, tetra-t-butoxytitanium, tri-iso-propoxy gallium, tri-iso-propoxy antimony,tri-iso-butoxy antimony, trimethoxy boron, triethoxy boron,tri-iso-propoxy boron, tri-n-propoxy boron, tri-iso-butoxy boron,tri-n-butoxy boron, tri-sec-butoxy boron, tri-t-butoxy boron,tri-iso-propoxy gallium, tetramethoxy germanium, tetraethoxy germanium,tetra-iso-propoxy germanium, tetra-n-propoxy germanium, tetra-iso-butoxygermanium, tetra-n-butoxy germanium, tetra-sec-butoxy germanium andtetra-t-butoxy germanium; halogenated compound such as antimonypentachloride, zinc chloride, lithium bromide, tin(IV) chloride, cadmiumchloride and boron trifluoride diethyl ether; alkyl aluminum such astrimethyl aluminum, triethyl aluminum, diethyl aluminum chloride, ethylaluminum dichloride and tri-iso-butyl aluminum; alkyl zinc such asdimethyl zinc, diethyl zinc and diisopropyl zinc; tertiary amines suchas triallylamine, triethylamine, tri-n-octylamine andbenzyldimethylamine; heteropolyacids such as phosphotungstic acid,phosphomolybdic acid, silicotungstic acid and alkali metal salt thereof;zirconium compounds such as zirconium acid chloride, zirconiumoctanoate, zirconium stearate and zirconium nitrate. More particularly,the catalyst is zirconium octanoate, tetraalkoxy zirconium or atrialkoxy aluminum compound.

Other ROP catalysts include metal-free organocatalysts that can providea platform to polymers having controlled, predictable molecular weightsand narrow polydispersities. Examples of organocatalysts for ringopening polymerization of cyclic esters, carbonates and siloxanes are4-dimethylaminopyridine, phosphines, N-heterocyclic carbenes (NHC),bifunctional aminothioureas, phosphazenes, amidines, and guanidines. Inan embodiment the catalyst isN-(3,5-trifluoromethyl)phenyl-N′-cyclohexyl-thiourea (TU):

In another embodiment, the catalyst and the accelerator are the samecompound, such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Anothermetal-free ROP catalyst comprises at least one1,1,1,3,3,3-hexafluoropropan-2-ol-2-yl (HFP) group. Singly-donatinghydrogen bond catalysts have the formula (15):R²—C(CF₃)₂OH  (15).R² represents a hydrogen or a monovalent radical having from 1 to 20carbons, for example an alkyl group, substituted alkyl group, cycloalkylgroup, substituted cycloalkyl group, heterocycloalkyl group, substitutedheterocycloalklyl group, aryl group, substituted aryl group, or acombination thereof. Exemplary singly-donating hydrogen bondingcatalysts are listed in Table 2.

TABLE 2

Doubly-donating hydrogen bonding catalysts have two HFP groups,represented by the general formula (16):

wherein R³ is a divalent radical bridging group containing from 1 to 20carbons, such as an alkylene group, a substituted alkylene group, acycloalkylene group, a substituted cycloalkylene group, aheterocycloalkylene group, substituted heterocycloalkylene group, anarylene group, a substituted arylene group, or a combination thereof.Representative double hydrogen bonding catalysts of formula (16) includethose listed in Table 3. In a specific embodiment, R² is an arylene orsubstituted arylene group, and the HFP groups occupy positions meta toeach other on the aromatic ring.

TABLE 3

In one embodiment, the catalyst is selected from the group consisting of4-HFA-St, 4-HFA-Tol, HFTB, NFTB, HPIP, 3,5-HFA-MA, 3,5-HFA-St, 1,3-HFAB,1,4-HFAB, and combinations thereof.

Also contemplated are catalysts comprising HFP-containing groups boundto a support. In one embodiment, the support comprises a polymer, acrosslinked polymer bead, an inorganic particle, or a metallic particle.HFP-containing polymers can be formed by known methods including directpolymerization of an HFP-containing monomer (for example, themethacrylate monomer 3,5-HFA-MA or the styryl monomer 3,5-HFA-St).Functional groups in HFP-containing monomers that can undergo directpolymerization (or polymerization with a comonomer) include acrylate,methacrylate, alpha, alpha, alpha-trifluoromethacrylate,alpha-halomethacrylate, acrylamido, methacrylamido, norbornene, vinyl,vinyl ether, and other groups known in the art. Typical examples of suchpolymerizeable HFP-containing monomers may be found in: Ito et al.,Polym. Adv. Technol. 2006, 17 (2), 104-115, Ito et al., Adv. Polym. Sci.2005, 172, 37-245, Ito et al., US20060292485, Maeda et al. WO2005098541,Allen et al. US20070254235, and Miyazawa et al. WO2005005370.Alternatively, pre-formed polymers and other solid support surfaces canbe modified by chemically bonding an HFP-containing group to the polymeror support via a linking group. Examples of such polymers or supportsare referenced in M. R. Buchmeiser, ed. “Polymeric Materials in OrganicSynthesis and Catalysis,” Wiley-VCH, 2003, M. Delgado and K. D. Janda“Polymeric Supports for Solid Phase Organic Synthesis,” Curr. Org. Chem.2002, 6 (12), 1031-1043, A. R. Vaino and K. D. Janda “Solid PhaseOrganic Synthesis: A Critical Understanding of the Resin”, J. Comb.Chem. 2000, 2 (6), 579-596, D. C. Sherrington “Polymer-supportedReagents, Catalysts, and Sorbents: Evolution and Exploitation—APersonalized View,” J. Polym. Sci. A. Polym. Chem. 2001, 39 (14),2364-2377, and T. J. Dickerson et al. “Soluble Polymers as Scaffold forRecoverable Catalysts and Reagents,” Chem. Rev. 2002, 102 (10),3325-3343. Examples of linking groups include C₁-C₁₂ alkyl, C₁-C₁₂heteroalkyl, an ether group, a thioether group, an amino group, an estergroup, an amide group, or a combination thereof. Also contemplated arecatalysts comprising charged HFP-containing groups bound by ionicassociation to oppositely charged sites on a polymer or a supportsurface.

The ROP reaction mixture comprises at least one catalyst and, whenappropriate, several catalysts together. The ROP catalyst is added in aproportion of 1/20 to 1/40,000 moles relative to the cyclic carbonylmonomers, and preferably of 1/100 to 1/20,000 moles.

The ring-opening polymerization is generally conducted in the presenceof an accelerator, in particular a nitrogen base. Exemplary nitrogenbase accelerators are listed below and include pyridine (Py),N,N-dimethylaminocyclohexane (Me₂NCy), 4-N,N-dimethylaminopyridine(DMAP), trans 1,2-bis(dimethylamino)cyclohexane (TMCHD),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (−)-sparteine, (Sp)1,3-bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1),1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2),1,3-bis(2,6-di-i-propylphenyl(imidazol-2-ylidene (Im-3),1,3-bis(1-adamantyl)imidazol-2-ylidene (Im-4),1,3-di-i-propylimidazol-2-ylidene (Im-5),1,3-di-t-butylimidazol-2-ylidene (Im-6),1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-7),1,3-bis(2,6-di-i-propylphenyl)-4,5-dihydroimidazol-2-ylidene,1,3-bis(2,6-di-i-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8) or acombination thereof, shown in Table 4.

TABLE 4

(Im-1)

(Im-2)

(Im-3)

(Im-4)

(Im-5)

(Im-6)

(Im-7)

(Im-8)

In an embodiment, the accelerator has two or three nitrogens, eachcapable of participating as a Lewis base, as for example in thestructure (−)-sparteine. Stronger bases generally improve thepolymerization rate.

The ROP reaction mixture also comprises an initiator. Initiatorsgenerally include nucleophiles such as alcohols, amines and thiols. Theinitiator can be monofunctional, difunctional, or multifunctional. Thenanostructure, as well as the surface modified nanoparticle comprisingthree or more peripheral ROP initiator groups, are examples ofmultifunctional initiators.

More particularly, the initiator for the ring opening reaction is analcohol. When a ROP polymer is prepared prior to attachment to thenanostructure, the alcohol initiator can be any suitable alcohol,including mono-alcohol, diol, triol, or other polyol, with the provisothat the choice of alcohol does not adversely affect the polymerizationyield, polymer molecular weight, complexation with a bio-activematerial, and/or the desirable mechanical and physical properties of thesurface modified nanoparticle. The alcohol can be multi-functionalcomprising, in addition to one or more hydroxyl groups, a halide, anether group, an ester group, an amide group, or other functional group.Exemplary alcohols includes methanol, ethanol, propanol, butanol,pentanol, amyl alcohol, capryl alcohol, nonyl alcohol, decyl alcohol,undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol,pentadecyl alcohol, cetyl alcohol, heptadecyl alcohol, stearyl alcohol,nonadecyl alcohol and other aliphatic saturated alcohols, cyclopentanol,cyclohexanol, cycloheptanol, cyclooctanol and other aliphatic cyclicalcohols; phenol, substituted phenols, benzyl alcohol, substitutedbenzyl alcohol, benzenedimethanol, trimethylolpropane, a saccharide,poly(ethylene glycol), propylene glycol, alcohol functionalized blockcopolymers derived from oligomeric alcohols, alcohol functionalizedbranched polymers derived from branched alcohols, or a combinationthereof. Monomeric diol initiators include ethylene glycols, propyleneglycols, hydroquinones, and resorcinols. An example of a diol initiatoris BnMPA, derived from 2,2-dimethylol propionic acid.

BnMPA is a precursor used in the preparation of cyclic carbonatemonomers.

The ring-opening polymerization can be performed with or without the useof a solvent, more particularly with a solvent. Optional solventsinclude dichloromethane, chloroform, benzene, toluene, xylene,chlorobenzene, dichlorobenzene, benzotrifluoride, petroleum ether,acetonitrile, pentane, hexane, heptane, 2,2,4-trimethylpentane,cyclohexane, diethyl ether, t-butyl methyl ether, diisopropyl ether,dioxane, tetrahydrofuran, or a combination comprising one of theforegoing solvents. When a solvent is present, a suitable cycliccarbonyl monomer concentration is about 0.1 to 5 moles per liter, andmore particularly about 0.2 to 4 moles per liter. In a specificembodiment, the reaction mixture for the ring-opening polymerization isfree of a solvent.

The ring-opening polymerization can be performed at a temperature thatis about ambient temperature or higher, more specifically a temperaturefrom 15° C. to 200° C., and more particularly 20° C. to 200° C. When thereaction is conducted in bulk, the polymerization is performed at atemperature of 50° C. or higher, and more particularly 100° C. to 200°C. Reaction times vary with solvent, temperature, agitation rate,pressure, and equipment, but in general the polymerizations are completewithin 1 to 100 hours.

Whether performed in solution or in bulk, the polymerizations areconducted in an inert (i.e., dry) atmosphere and at a pressure of from100 to 500 MPa (1 to 5 atm), more typically at a pressure of 100 to 200MPa (1 to 2 atm). At the completion of the reaction, the solvent can beremoved using reduced pressure.

The nitrogen base accelerator is present in an amount of 0.1 to 5.0 mol%, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2 to 0.5 mol %, based ontotal moles of cyclic carbonyl monomer.

The amount of initiator is calculated based on the equivalent molecularweight per nucleophilic initiating group in the initiator (e.g., alcoholgroups). The initiating groups are present in an amount of 0.001 to 10.0mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, and 0.2 to 0.5 mol %, basedon total moles of cyclic carbonyl monomer. For example, if the molecularweight of the initiator is 100 g/mole and the initiator has 2 hydroxylgroups, the equivalent molecular weight per hydroxyl group is 50 g/mole.If the polymerization calls for 5 mol % hydroxyl groups per mole ofmonomer, the amount of initiator is 0.05×50=2.5 g per mole of monomer.

In a specific embodiment, the catalyst is present in an amount of about0.2 to 20 mol %, the nitrogen base accelerator is present in an amountof 0.1 to 5.0 mol %, and the hydroxyl groups of the initiator arepresent in an amount of 0.1 to 5.0 mol % based on the equivalentmolecular weight per nucleophilic group in the initiator.

As stated above, the ring opening polymerization forms a polymer chaincomprising a living polymer segment. In an embodiment, one backbonerepeating unit of the ROP polymer chain is a carbonate repeating unit.The ROP polymer backbone can, for example, comprise a polyesterhomopolymer, a random polyester copolymer, a polycarbonate homopolymer,a random polycarbonate copolymer, or a random polyestercarbonatecopolymer. The ROP polymer chain can comprise a terminal hydroxyl group,terminal thiol group, or terminal amine group, each of which caninitiate further ROP chain growth, if desired.

The ROP polymer can comprise hydrophilic repeat units, hydrophobicrepeat units, and combinations thereof, thereby imparting amphiphilicproperties to the surface modified nanoparticles. The ROP polymer chainscan have a number average molecular weight M_(n) as determined by sizeexclusion chromatography of at least 2500 g/mol, more specifically 4000g/mol to 150000 g/mol, and even more specifically 10000 g/mol to 50000g/mol. In an embodiment, the ROP polymer chain has a number averagemolecular weight M_(n) of 10000 to 20000 g/mole. The ROP polymer chainsalso have a narrow polydispersity index (PDI), generally from 1.01 to1.35, more particularly 1.1 to 1.30, and even more particularly 1.1 to1.25.

The catalysts can be removed by selective precipitation, or in the caseof the solid supported catalysts, simply by filtration. The ROP polymercan comprise residual catalyst in an amount greater than 0 wt. %, basedon total weight of the first polymer and the residual catalyst. Theamount of residual catalyst can also be less than 20 wt. %, less than 15wt. %, less than 10 wt. %, less than 5 wt. %, less than 1 wt. %, or mostspecifically less than 0.5 wt. % based on the total weight of the firstpolymer and the residual catalyst.

As stated above, the ROP polymer can comprise a pendant latentcarboxylic acid group, such as a benzyl ester. In this instance, thelatent carboxylic acid group can be deprotected using H₂/Pd—C to form apendant carboxylic acid group. If the protected carboxylic acid is inthe form of a thermally labile carboxylic ester, such as a t-butylester, deprotection can be effected by heating the ROP polymer. If theprotected carboxylic acid is hydrolytically unstable, such as atrifluoroethyl ester, the ROP polymer can be deprotected with mildaqueous acid or base to form a pendant carboxylic acid group. In aparticular embodiment, the protected carboxylic acid is a benzyl ester.

Also disclosed are methods of forming compositions comprising thesurface modified nanoparticles. One method comprises independentlycovalently attaching by a ring opening reaction a first cyclic carbonylmonomer independently to three or more nucleophilic surface groups of ananostructure, thereby forming a first modified nanoparticle, the firstmodified nanoparticle comprising three or more initiator groups producedby the ring opening of the first cyclic carbonyl monomer; andindependently initiating a ring opening polymerization of a mixturecomprising one or more cyclic carbonyl monomers by the three or moreinitiator groups, thereby forming a surface modified nanoparticlecomprising three or more independent surface branches, wherein each ofthe three or more independent surface branches comprises a first polymerproduced by the ring opening polymerization; wherein the first cycliccarbonyl monomer has the general formula (12):

wherein each Y is independently selected from the group consisting of—O—, —NH—,

and —S—, R⁵ and R⁶ are independent monovalent radicals selected from thegroup consisting of hydrogen and monovalent hydrocarbon radicalscomprising 1 to 30 carbons, and M¹ is a monovalent radical selected fromthe group consisting of —OR¹, —NHR¹, —NR¹R¹, and —SR¹, wherein R¹ is amonovalent radical. In an embodiment, the first cyclic carbonyl monomeris a cyclic carbonate, and M¹ comprises a second polymer. In anotherembodiment, the second polymer is a poly(alkylene ether). In anotherembodiment, the first cyclic carbonyl monomer does not comprise apolymeric substituent. In another embodiment, one or more of the one ormore cyclic carbonyl monomers comprises a functional group comprising apoly(alkylene ether). In another embodiment, each of the three or moreindependent surface branches comprises a repeat unit comprising a sidechain comprising a monovalent leaving group, and the method furthercomprises treating the surface modified nanoparticle with a tertiaryamine, thereby forming a second surface modified nanoparticle comprisinga quaternary amine. In another embodiment, the first polymer is a blockcopolymer. In another embodiment, one or more of the one or more cycliccarbonyl monomers comprises a functional group selected from the groupconsisting of latent carboxylic acid groups, functional groups capableof reacting with a tertiary amine to form a quaternary amine, ureagroups, polyether groups, and combinations thereof. In anotherembodiment, the nanostructure is a macromolecule selected from the groupconsisting of crosslinked polymers, latexes, hyperbranched polymers,dendrimers, dendrons, cyclodextrins, and combinations of the foregoingmaterials. In another embodiment, the nanostructure comprises aninorganic material selected from the group consisting of cobalt, iron,and combinations thereof. In another embodiment, the three or morenucleophilic surface groups are terminal amine groups, and thenanostructure is a polypropyleneimine dendrimer selected from the groupconsisting of DAB4, DAB8, DAB16, DAB32 and DAB64.

The above described method can further comprise forming a first aqueousmixture comprising the surface modified nanoparticle, and contacting thefirst aqueous mixture with a second aqueous mixture comprising abiologically active material, thereby forming a loaded nanoparticle. Themethod can further comprise contacting a cell with the loadednanoparticle. Another method of preparing a loaded nanoparticlecomprises contacting a first aqueous mixture comprising the abovedescribed surface modified nanoparticles with a second aqueous mixturecomprising a biologically active material. In an embodiment, thebiologically active material is a gene or a drug.

Another method related to forming a surface modified nanoparticlecomprises independently attaching by a ring opening reaction a firstcyclic carbonyl monomer to three or more nucleophilic surface groups ofa nanostructure, thereby forming a first modified nanoparticle, thefirst modified nanoparticle comprising three or more functional groupsF′ and three or more initiator groups; and independently initiating byeach of the three or more initiator groups a ring opening polymerizationof one or more cyclic carbonyl monomers, thereby forming a surfacemodified nanoparticle comprising three or more independent surfacebranches, wherein each of the three or more independent surface brancheshas the general formula (3):

wherein

X^(d) is a divalent radical selected from the group consisting of —O—,

—S—, and combinations thereof, R⁴ is a monovalent radical comprising 1to 30 carbons,

j is an integer greater than or equal to 1,

P′ is comprises a first polymer comprising a backbone selected from thegroup consisting of polycarbonates, polyesters, polyureas,polycarbamates, polythiocarbamates, polythioureas, and combinationsthereof,

each X^(e), X^(f), and X^(g) is independently selected from the groupconsisting of —O—,

—S—, and combinations thereof, wherein R⁶ is hydrogen or a monovalenthydrocarbon radical comprising 1 to 30 carbons,

each R⁵ is an independent monovalent radical selected from the groupconsisting of hydrogen and monovalent hydrocarbon radicals comprising 1to 30 carbons, and

each functional group F′ is independently a monovalent radical.

The ROP polymer chains of the surface modified nanoparticles cancomprise repeat units comprising a positive charge, a negative charge,or a mixture thereof. The ROP polymer chains can comprise from 1 to 250mmoles carboxylic acid per gram of nanoparticles, more particularly morethan 3 to 50 mmoles carboxylic acid per gram of nanoparticles, and evenmore particularly 3 to 40 mmoles carboxylic acid per gram ofnanoparticles. The ROP polymer chains can comprise 1 to 250 mmoles of aurea functional group per gram of nanoparticles, more particularly 3 to50 mmoles of a urea functional group per gram of nanoparticles, and evenmore particularly 3 to 40 mmoles of a urea functional group per gram ofnanoparticles.

In aqueous solution the surface modified nanoparticles have an averageparticle size of from 2 nm to 500 nm, 10 nm to 250 nm, and moreparticularly 50 nm to 200 nm, 50 nm to 150 nm, 50 nm to 120 nm, and evenmore particularly from 50 nm to 100 nm, as measured by dynamic lightscattering (Brookhaven Instrument Corp., Holtsville, N.Y., U.S.A.)equipped with a He—Ne laser beam at 658 nm (scattering angle: 90°). Theparticle size measurements are repeated for 5 runs for each sample, andthe particle size are reported as the average of 5 readings. For theforegoing particle sizes, the aqueous solution can have a pH of from 5.0to 8.0.

Also disclosed are modified nanoparticles loaded with biologicallyactive cargo materials, such as a gene, a nucleotide, a protein, apeptide, a drug, or combinations thereof. These are referred to hereinas loaded nanoparticles. In aqueous solution at a pH of from 5.0 to 8.0,the loaded nanoparticles have an average particle size of from 2 nm to500 nm, 2 nm to 250 nm, 2 nm to 150 nm, 2 nm to 120 nm, and moreparticularly 10 nm to 120 nm, 20 nm to 120 nm, 30 nm to 120 nm, and evenmore particularly from 50 nm to 120 nm, as measured by dynamic lightscattering (Brookhaven Instrument Corp., Holtsville, N.Y., U.S.A.)equipped with a He—Ne laser beam at 658 nm (scattering angle: 90°). Theparticle size measurements are repeated for 5 runs for each sample, andthe particle size are reported as the average of 5 readings. The loadednanoparticles can comprise, for example 0.1 to 90 wt. %, moreparticularly 5 to 50 wt. %, and even more particularly 15 to 50 wt. % ofa biologically active material based on total dry weight of the loadednanoparticles. In an embodiment, the biologically active cargo materialis a drug.

Also disclosed is a method of preparing a loaded nanoparticle fortreating a cell, comprising contacting a first aqueous mixturecomprising a modified nanoparticle comprising a surface branchcomprising a ROP polymer chain derived by ring opening polymerization ofone or more cyclic carbonyl monomers, with a second aqueous mixturecomprising a biologically active cargo material, thereby forming a thirdmixture comprising the loaded nanoparticle; wherein the loadednanoparticle has a particle size of 10 nm to 500 nm at a pH of from 5.0to 8.0.

Further disclosed is a method of treating a cell, comprising contactingthe cell with an aqueous mixture comprising the above described loadednanoparticles. The biologically active cargo can comprise a singlebiologically active material or a mixture of biologically activematerials. The biologically active cargo can be a drug, for exampledoxorubicin. Cells can be contacted in vitro, ex vivo, or in vivo.Contacting induces 0% to 20%, 0% to 15%, 0% to 10%, 0% to 5%, 0% to 2%,or more particularly 0% to 1% cytotoxicity. In an embodiment, contactinginduces no cytotoxicity.

The present loaded nanoparticles can comprise both small molecularweight biologically active materials in the size range from 100 daltonsto about 1,000 daltons as well as larger macromolecular materials, suchas peptide and protein drugs in the size range from about 1,000 daltonsto about 100,000 daltons, and beyond. Exemplary protein drugs includepeptide hormones such as insulin, glucagon, parathyroid hormone,calcitonin, vasopression, renin, prolactin, growth hormone, thegonadotropins including chorionic gonadotropin, follicle stimulatinghormone, thyroid stimulating hormone and leutenizing hormone;physiologically active enzymes such as transferases, hydrolases, lyases,isomerases, phosphatases, glycosidases, superoxide dismutase, factorVIII, plasminogen activators; and other therapeutic agents includingprotein factors such as epidermal growth factor, insulin-like growthfactor, tumour necrosis factor, transforming growth factors, fibroblastgrowth factors, patelet-derived growth factors, erythropoietin, colonystimulating factors, bone morphogenetic proteins, interleukins andinterferons. Exemplary non-protein macromolecules includepolysaccharides, nucleic acid polymers, and therapeutic secondarymetabolites including plant products such as vinblastine, vincristine,taxol and the like.

Other exemplary drugs include Aspirin, Diflunisal, Diclofenac,Aceclofenac, Acemetacin, Etodolac, Indometacin, Sulindac, Tolmetin,Ibuprofen, Carprofen, Fenbufen, Fenoprofen, Flurbiprofen, Ketoprofen,Ketorolac, Loxoprofen, Naproxen, Oxaprozin, Tiaprofenic acid, Suprofen,Mefenamic acid, Meclofenamic acid, Lumiracoxib, Oxyphenbutazone,Piroxicam, Lornoxicam, Meloxicam, and Tenoxicam. SteroidalAnti-Inflammatory Drugs include Hydrocortisone, Prednisone,Prednisolone, Methylprednisolone, Dexamethasone, Betamethasone,Triamcinolone, Beclometasone, Fludrocortisone acetate, and Aldosterone.Chemotherapeutic drugs include Doxorubicin and DNA alkylating Agentssuch as Melphalan, Chlorambucil, Dacarbazine, Temozolomide, andStreptozotocin. Antimetabolite drugs include Methotrexate, Pemetrexed,Raltitrexed, Tioguanine, Fludarabine, Pentostatin, Cladribine,Floxuridine, and Gemcitabine. Alkaloid drugs include Vincristine,Vinblastine, Vinorelbine, Vindesine, and Topoisomerase. Inhibitorsinclude Etoposide, Teniposide, Irinotecan, and Topotecan. Taxanesinclude Paclitaxel and Docetaxel. Anticoagulants include Warfarin,Acenocoumarol, Phenprocoumon, Argatroban, and Ximelagatran.

Still other exemplary commercially available drugs include13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine,5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine,Abraxane, Accutane®, Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®,Agrylin®, Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin,Alkaban-AQ®, Alkeran®, All-transretinoic Acid, Alpha Interferon,Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide,Anandron®, Anastrozole, Arabinosylcytosine, Ara-C, Aranesp®, Aredia®,Arimidex®, Aromasin®, Arranon®, Arsenic Trioxide, Asparaginase, ATRA,Avastin®, Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene,BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib,Busulfan, Busulfex®, C225, Calcium Leucovorin, Campath®, Camptosar®,Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine,Carmustine Wafer, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU,Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor,Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®,Cytarabine, Cytarabine Liposomal, Cytosar-U®, Cytoxan®, Dacarbazine,Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin,Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal,DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, DenileukinDiftitox, DepoCyt™, Dexamethasone, Dexamethasone Acetate, DexamethasoneSodium Phosphate Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,Doxil®, Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®,Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™, Elspar®, Emcyt®,Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-asparaginase,Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide Phosphate,Eulexin®, Everolimus, Evista®, Exemestane, Fareston®, Faslodex®,Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®,Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, FolinicAcid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumabozogamicin, Gemzar, Gleevec™, Gliadel® Wafer, GM-CSF, Goserelin,Granulocyte—Colony Stimulating Factor, Granulocyte Macrophage ColonyStimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexalen®,Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®,Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone SodiumSuccinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, IbritumomabTiuxetan Idamycin®, Idarubicin, Ifex®, IFN-alpha Ifosfamide, IL-11 IL-2Imatinib mesylate, Imidazole Carboxamide Interferon alfa, InterferonAlfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A®(interferon alfa-2b), Iressa®, Irinotecan, Isotretinoin, Ixabepilone,Ixempra™, K Kidrolase (t), Lanacort®, Lapatinib, L-asparaginase, LCR,Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine™, Leuprolide,Leurocristine, Leustatin™, Liposomal Ara-C, Liquid Pred®, Lomustine,L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®, Matulane®, Maxidex,Mechlorethamine, Mechlorethamine Hydrochloride, Medralone®, Medrol®,Megace®, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna,Mesnex™, Methotrexate, Methotrexate Sodium, Methylprednisolone,Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC,MTX, Mustargen®, Mustine Mutamycin®, Myleran®, Mylocel™, Mylotarg®,Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®,Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®,Novantrone®, Octreotide, Octreotide acetate, Oncospar®, Oncovin®,Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel,Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin®,Paraplatin®, Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim,PEG-INTRON™, PEG-L-asparaginase, PEMETREXED, Pentostatin, PhenylalanineMustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a) Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, and Zometa.

No restriction is placed on the type of cell that can be treated withthe above-described loaded nanoparticles. In particular, the cells canbe eukaryotic cells, mammalian cells, and more particularly rodent orhuman cells. The cells can be derived from various tissues, includingextraembryonic or embryonic stem cells, totipotent or pluripotent,dividing or non-dividing, parenchyma or epithelium, immortalized ortransformed, or the like. The cell may be a stem cell or adifferentiated cell. Cell types that are differentiated includeadipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,dendritic cells, neurons, glia, mast cells, blood cells and leukocytes(e.g., erythrocytes, megakaryotes, lymphocytes, such as B, T and naturalkiller cells, macrophages, neutrophils, eosinophils, basophils,platelets, granulocytes), epithelial cells, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine orexocrine glands, as well as sensory cells.

The above-described loaded nanoparticles can be used as non-viraltransfection vectors. The target gene is not limited to any particulartype of target gene or nucleotide sequence. For example, the target genecan be a cellular gene, an endogenous gene, an oncogene, a transgene, ora viral gene including translated and non-translated RNAs. Exemplarypossible target genes include: transcription factors and developmentalgenes (e.g., adhesion molecules, cyclin-dependent kinase inhibitors, Wntfamily members, Pax family members, Winged helix family members, Hoxfamily members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2,CBL, CSFIR, ERBA, ERBB, ERBB2, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS,JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIMI, PML,RET, SKP2, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g., APC,BRAI, BRCA2, CTMP, MADH4, MCC, NFI, NF2, RBI, TP53, and WTI); andenzymes (e.g., ACP desaturases and hydroxylases, ADP-glucosepyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cyclooxygenases, decarboxylases,dextrinases, DNA and RNA polymerases, galactosidases, glucose oxidases,GTPases, helicases, integrases, insulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, peroxidases,phosphatases, phospholipases, phosphorylases, proteinases andpeptidases, recombinases, reverse transcriptases, telomerase, includingRNA and/or protein components, and topoisomerases).

Summarizing, surface modified nanoparticles have been describedcomprising surface branches comprising an ROP polymer chain derived fromring opening reaction of one or more cyclic carbonyl monomers. The ROPpolymer chain can comprise a wide variety of functional groups such asbiodegradable urea, quaternary amine, and/or carboxylic acid containinggroups, which provide versatility in tailoring the binding strength andrelease properties of the loaded nanoparticles. The biologically activecargo can be of diverse size and functionality. The loaded nanoparticlescan potentially be useful for delivery of small molecular drugs andproteins, and/or for simultaneous delivery of drugs and genes, or drugsand proteins.

The preparation and use of the surface modified nanoparticles based onthe above-described ring opening methods is further illustrated by thefollowing examples.

EXAMPLES

Part 1. Convergent Approach—Small Stars

Example 1 Model Reaction. Formation of MTCOMPEG, a MPEG Functionalizedwith a Cyclic Carbonate

MPEG (M_(n) 2000 g/mol, n˜44, PDI 1.02, 10 g, 5 mmol, 1 eq.,) wasdissolved in tetrahydrofuran (THF) (20 mL, 2.5 M) by heating themixture. MTCOPFP (MW 326 g/mol, 3.61 g, 11 mmol, 2.2 eq.) and cesiumfluoride (CsF) (MW 152 g/mol, 0.34 g, 2 mmol, 0.4 eq.) were added. After48 hours THF was evaporated. The crude product was purified first byextraction in a mixture of dichloromethane (500 mL) and hydrochloricacid (500 mL, 0.1 M) and second by polymer precipitation in cold ethylether. The product MTCOMPEG was dried in vacuum until a constant weightwas achieved. M_(n)=2159 g/mol, PDI=1.04. ¹H NMR (400 MHz, CDCl₃):delta=4.69 (d, 8H, 4×CH₂OCOO), 4.31 (m, 8H, 4×PEG-CH—OCO), 4.19 (d, 8H,4×CH₂OCOO), 3.84-3.44 (m, poly, 4×OCH ₂CH _(2 PEG)+end groups), 3.37 (s,12H, 4×OCH₃), 1.33 (s, 12H, 4×CH₃). ¹³C NMR (400 MHz, CDCl₃):delta=171.0, 147.3, 72.8, 71.8, 70.8, 70.4, 68.6, 64.8, 59.0, 40.1,30.8, 17.5

Example 2 Model Reaction. Benzylamine Ring Opening of the CyclicCarbonate MTCOMPEG

MTCOMPEG (M_(n) 2000 g/mol, n˜44, PDI 1.04, 0.5 g, 0.25 mmol, 1 eq.) wasdissolved in THF (1 mL, 0.25 M) with heating. When the solution reachedroom temperature benzyl amine (MW 107 g/mol, 0.027 g, 0.25 mmol, 1 eq.)was added. The reaction was heated to 40° C., stirred overnight, andprecipitated in cold ethyl ether. The product MPEGB was dried in vacuumuntil a constant weight was achieved. M_(n)=2250 g/mol, PDI=1.04. ¹H NMR(400 MHz, CDCl₃): delta=7.28 (m, 5H, Ar), 4.34-4.22 (m, 6H, CH₂—OH+CH₂—OCO+Ar—CH ₂—NHCO), 3.80-3.40 (m, poly, OCH ₂CH _(2 PEG)+endgroups), 3.35 (s, 3H, OCH₃), 1.17 (s, 3H, CH₃). ¹³C NMR (400 MHz,CDCl₃): delta=174.2, 156.8, 138.3, 128.6, 127.4, 72.8, 70.8, 70.4, 68.7,66.0, 64.5, 63.4, 58.9, 48.8, 44.9, 17.3

Example 3 MPEGB Reacted with Reacted with Acetyl Chloride to FormMPEGBAc

MPEGB (M_(n) 2250 g/mol, PDI 1.04, 0.050 g, 0.03 mmol, 1 equi) wasdissolved in dichloromethane (DCM) (1 mL, 0.03 M). Acetyl chloride(M_(n) 79 g/mol, 0.002 g, 0.03 mmol, 1.2 eq.) and triethylamine (TEA)(M_(n) 101 g/mol, 0.003 g, 0.03 mmol, 1.3 eq.) were added. After 4hours, the solution was extracted against water. The solvent wasevaporated and the product was dried in vacuum until a constant weightwas achieved. ¹H NMR (400 MHz, CDCl₃): delta=7.28 (m, 5H, Ar), 4.34-4.16(m, 4H, CH₂—OCO+Ar—CH ₂—NHCO), 3.84-3.42 (m, poly, OCH ₂CH _(2 PEG)+endgroups), 3.35 (s, 3H, OCH₃), 2.05 (s, 2H, CH₂—OCO), 1.17 (s, 3H, CH₃).¹³C NMR (400 MHz, CDCl₃): δ=174.2, 156.7, 138.3, 128.7, 127.5, 71.8,70.4, 68.8, 66.0, 64.5, 63.5, 58.9, 48.8, 44.9, 29.5, 17.3.

The above reaction models an alternative approach to constructingsurface branches on nanostructures comprising three or more nucleophilicsurface groups. In this approach, a first cyclic carbonyl monomercomprises a pendant ROP polymer (or another polymer such as apolyethylene glycol). The first cyclic carbonyl monomer is attachedindependently in a ring opening reaction to each of the three or morenucleophilic groups of a nanostructure. The initiator groups formed bythe ring opening reaction (e.g., hydroxyl group) are not used toinitiate a ring opening polymerization in a subsequent step. Instead,the initiator groups are derivatized with other useful functionalmoieties, either non-polymeric (e.g., an acetate group) or polymeric(e.g., reactive pre-formed ROP polymer or other polymer). This approachis less preferred due to steric constraints in attaching large groups tothe initiator group formed by the first ring opening reaction.

Stars.

Example 4 Ring Opening of the Cyclic Carbonate MTCOMPEG withPolypropylenimine Tetraamine Dendrimer Generation 1 (DAB4) to FormTetrol D4MPEG

MTCOMPEG (M_(n) 2000 g/mol, n˜44, PDI 1.04, 1.0 g, 0.50 mmol, 4 eq.) wasdissolved in THF (2 mL, 0.25 M) with heating. When the solution hadreached room temperature DAB4 (M_(n) 316 g/mol, 0.040 g, 0.13 mmol, 1eq.) was added. After 40 hours the crude product was precipitated incold ethyl ether and dried under vacuum. The product was thenfractionally precipitated with dichloromethane (6 mL) as a good solvent,2-propanol (20 mL) as semi-good solvent and ethyl ether (60 mL) asnon-solvent. The product D4MPEG was filtered and dried under vacuumuntil a constant weight was achieved. M_(n)=8 326 g/mol, PDI=1.08. ¹HNMR (400 MHz, CDCl₃): δ=5.95 (b, 4H, 4×NH—OCO), 4.30-4.10 (m, 16H, 8×CH₂—OCO), 3.83-3.42 (m, poly, 4×OCH ₂CH _(2 PEG)+end groups), 3.73 (s, 8H,4×CH ₂—OH), 3.37 (s, 12H, 4×OCH₃), 3.18 (b, 8H, 4×CH ₂—NH—OCO), 2.41 (b,8H, 4×CH₂—N), 2.38 (b, 4H, 2×CH₂—N), 1.62 (b, 8H, 4×CH₂), 1.42 (s, 12H,4×CH₃), 1.24 (b, 4H, 2×CH ₂—CH₂—N). ¹³C NMR (400 MHz, CDCl₃): δ=71.8,70.4, 68.8, 65.6, 64.2, 63.5, 58.9, 48.7, 39.8, 26.8, 25.2, 17.4.

In the following examples, the polymers are represented by the generalnotation:I′—[P(Monomer Mixture 1)_(n/q′)-b-P(Monomer Mixture 2)_(m/q′). . .etc.)]_(q′)wherein I′ represents a group derived from an initiator for ring openingpolymerization, q′ is the number of initiating sites on I′, n is thetotal moles of cylic carbonyl monomer in Monomer Mixture 1, m is thetotal moles of cylic carbonyl monomer in Monomer Mixture 2, and E′ is anend cap group when present. When the initiator is a polymer, it isunderstood that the initiator forms a block of a block copolymer, whichcan be indicated by insertion of “-b-” between the initiator name I′ andthe brackets. The brackets “[ ]” enclose the one or more polymer chainfragments formed by ring opening polymerization, “P( )” indicates apolymer formed by the one or more cyclic carbonyl monomers enclosed bythe parentheses, “-r” indicates random copolymer, and “-b-” indicates ablock boundary. For example, the polymer MPEG-b-[P(MTCOEt-r-MTCU)] hasthe following chemical structure:

MPEG-b-[P(MTCOEt-r-MTCU)] comprises a hydrophilic block derived from apolyether initiator, monomethyl poly(ethylene glycol) (MPEG), and ahydrophobic block comprising a random copolymer derived from two cycliccarbonyl monomers MTCOEt and MTCU. The vertical stacking of thecarbonate repeat units indicates random arrangement of the repeat units;that is, either carbonate repeat unit can be attached to the MPEG chain.The initiator and monomers have the following chemical structures.

Example 5 Ring Opening Polymerization of MTCOEt Initiated by D4MPEG,DP=8

D4MPEG was dried over CaH₂ in THF before being transferred to the glovebox. D4MPEG (M_(n) 8326 g/mol, PDI 1.08, 0.099 g, 0.01 mmol, 1 eq.) wasdissolved in dry dichloromethane (1.0 mL, 1 M). DBU (MW 152 g/mol, 0.004g, 0.02 mmol, 2 eq.) was added and reaction where stirred for 30 min. TU(M_(n) 370 g/mol, 0.009 g, 0.02 mmol, 2 eq.) and MTCOEt (M_(n) 188g/mol, 0.073 g, 0.39 mmol, 32 eq.) were then added. After 2 hours thereaction was quenched with benzoic acid (MW 102 g/mol, 0.021 g, 0.21mmol, 20 eq.) and stirred for 30 min before precipitation in ethylether. The product D4MPEG-[P(MTCOEt)]₄ was dried in vacuum until aconstant weight was achieved. PDI=1.09. ¹H NMR (400 MHz, CDCl₃):delta=7.28 (m, 5H, Ar), 4.27 (m, poly, C—CH₂—O_(polymer backbone)), 4.17(m, poly, CH₃—CH ₂—O_(poly(ethyl MTC))), 3.80-3.40 (m, poly, OCH ₂CH_(2 PEG)+end groups), 3.35 (s, 3H, OCH₃), 1.30-1.18 (m, poly,2×CH_(3 poly(ethyl MTC))+end groups). ¹³C NMR (400 MHz, CDCl₃):delta=172.0, 154.5, 135.8, 128.6, 127.4, 127.2, 125.4, 72.0, 70.4, 69.0,68.7, 68.5, 67.0, 64.5, 61.3, 61.0, 58.9, 47.0, 46.2, 34.2, 30.3, 28.6,25.7, 21.0, 17.3, 14.0.

Part 2. Divergent Approach—Small Stars

Example 6 Ring Opening of the Cyclic Carbonate MTCU Initiated by DAB4 toForm D4PEU

MTCU (MW 322 g/mol, 0.260 g, 0.81 mmol, 4.1 eq.) was dissolved in THF(10 mL, 0.08 M). DAB4 (MW 316 g/mol, 0.062 g, 0.20 mmol, 1 eq.) wasadded. After 72 hours calcium hydride was added. Another 24 hours laterthe mixture was filtrated, the solvent was evaporated, and the drytetrol compound D4PEU was transferred to a glovebox M_(n)=1604 g/mol. ¹HNMR (400 MHz, Acetone): delta=8.11 (s, 4H, 4×OC—NH—Ar), 7.46+7.20+6.91(d+t+t, 20H, 4×NH—Ar), 6.67 (t, 4H, 4×OC—NH), 6.10 (b, 4H, 4×OCC—NH),4.24-4.14 (m, 24H, 4×CH ₂—OH+8×CH ₂—OCO), 3.46 (m, 8H, 4×CH ₂—NH—CONH),3.14 (m, 8H, 4×CH ₂—NH—COO), 2.40 (m, 12H, 6×CH₂—N), 1.61 (m, 8H,4×CH₂), 1.42 (m, 4H, 2×CH₂), 1.15 (s, 12H, 4×CH₃).

Example 7 Ring Opening of the Cyclic Carbonate MTCOBn Initiated by DAB4to Form D4Bn

MTCOBn (MW 250 g/mol, 0.512 g, 2.05 mmol, 4.4 eq.) was dissolved in THF(10 mL, 0.08 M). DAB4 (MW 316 g/mol, 0.150 g, 0.47 mmol, 1 eq.) wasadded. After 72 hours calcium hydride was added. Another 24 hours laterthe mixture was filtrated, the solvent was evaporated and the drycompound D4Bn was transferred to a glovebox. M_(n)=1317 g/mol, PDI=1.04.¹H NMR (400 MHz, CDCl₃): δ=7.38 (b, 20H, 4×Ar), 5.90 (b, 4H, 4×OOC—NH),5.18 (s, 8H, 4×Ar—CH ₂—OCO), 4.38+4.21 (d+d, 16H, 4×CH ₂—OCO), 3.70 (m,8H, 4×CH ₂—OH) 3.20 (m, 8H, 4×CH ₂—NH—COO), 2.40 (m, 12H, 6×CH₂—N),1.70-1.60 (m, 12H, 6×CH₂), 1.20 (s, 12H, 4×CH₃).

Example 8 Ring Opening Polymerization of MTCOEt Initiated by D4Bn toForm D4Bn-[P(MTCOEt)]₄, Targeted Degree of Polymerization (DP)=15

D4Bn (MW 1317 g/mol, 0.101 g, 0.08 mmol, 1 eq.) was dissolved in drydichloromethane (2 mL, 2.3 M). DBU (MW 152 g/mol, 0.037 g, 0.24 mmol, 3eq.) was added and stirred for 30 minutes. TU (MW 370 g/mol, 0.084 g,0.23 mmol, 3 eq.) and MTCOEt (MW 188 g/mol, 0.861 g, 4.58 mmol, 60 eq.)was added. After 1.5 hours a portion of the reaction mixture wastransferred to a new flask to work as an initiator for the growth of anadditional block. The part that was not used for further reactions wasquenched with benzoic acid and stirred for 30 min before precipitationin 2-propanol. The product D4Bn-[P(MTCOEt)_(n/4)]₄ was dried in vacuumuntil a constant weight was achieved. M_(n)=12597 g/mol, PDI=1.37. ¹HNMR (400 MHz, CDCl₃): δ=7.32, (b, 20H, 4×Ar), 5.14 (s, 8H, 4×Ar—CH₂—OCO), 4.28 (m, poly, 4×C—CH₂—O_(polymer backbone)), 4.18 (m, poly,4×CH₃—CH ₂—O_(poly(ethyl MTC))), 3.70 (s, 8H, 4×C—CH ₂—OH), 1.30-1.16(m, poly, 2×CH_(3 poly(ethyl MTC))+end groups).

Example 9 Ring Opening Polymerization of a Mixture of MTCOMPEG(M_(n)=0.5 k) and MTCOEt (Molar Ratio x:y=1:4, Respectively) Initiatedby D4Bn-[P(MTCOEt)_(n/4)]₄, to FormD4Bn4-[P(MTCOEt)_(n/4)-b-P(MTCOMPEG_(x/4)-r-MTCOEt_(y/4))]

A portion of the reaction mixture of Example 8 in its solutioncontaining both DBU and TU was added to a new flask. The portioncontained D4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.200 g,0.02 mmol, 1 eq.). More DBU was added to obtain 0.1 eq. of DBU comparedto the monomers MTCOMPEG and MTCOEt. MTCOMPEG (M_(n) 659 g/mol, 0.170 g,0.26 mmol, 16 eq.) was mixed with MTCOEt (MW 188 g/mol, 0.202 g, 1.07mmol, 64 eq.) in DCM (2 mL). The solution of the monomers was mixed withthe initiator D4Bn-[P(MTCOEt)_(n/4)]₄. After 3 hours the reaction wasquenched with benzoic acid and stirred for 30 min. The productD4Bn-[P(MTCOEt)_(n/4)-b-P(MTCOMPEG_(x/4)-r-MTCOEt_(y/4))]₄ wasprecipitated in ethyl ether and dried in vacuum until a constant weightwas achieved. PDI=1.36 before dialysis. ¹H NMR (400 MHz, CDCl₃):delta=7.32, (b, 20H, 4×Ar), 5.14 (s, 8H, 4×Ar—CH₂—O), 4.28 (m, poly,4×C—CH₂—O_(polymer backbone)), 4.18 (m, poly,4×CH₃—CH₂—O_(poly(ethyl MTC))), 3.72-3.52 (m, poly, 4×OCH₂CH_(2 PEG)+endgroups), 3.38 (s, poly, OCH_(3 poly(PEG MTC))), 1.28-1.21 (m, poly,2×CH_(3 poly(ethyl MTC))+end groups).

Example 10 ROP Polymerization of a Mixture of MTCOMPEG (M_(n)=0.5 k) andMTCOEt (Molar Ratio x:y=2:3, Respectively) Initiated byD4Bn4-[P(MTCOEt)_(n/4)]₄, Target DP=20

A portion of Example 8 in its solution containing both DBU and TU wasadded to a new flask, as described for Example 9. The portion containedD4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.120 g, 0.01mmol, 1 eq.). More DBU was added to obtain 0.1 eq. of DBU compared tothe monomers. MTCOMPEG (M_(n) 659 g/mol, 0.200 g, 0.30 mmol, 32 eq.) wasmixed with MTCOEt (MW 188 g/mol, 0.086 g, 0.46 mmol, 48 eq.) indichloromethane (2 mL, 2.3 M). The solution of the monomers was mixedwith initiator D4Bn-[P(MTCOEt)_(n/4)]₄. After 3 hours the reaction wasquenched with benzoic acid and stirred for 30 min. The product wasprecipitated in ethyl ether and dried in vacuum until a constant weightwas achieved. ¹H NMR (400 MHz, CDCl₃): delta=7.32 (m, 20H, 4×Ar), 5.02(s, 8H, 4×Ar—CH₂—NH), 4.28 (m, poly, 4×C—CH₂—O_(polymer backbone)), 4.18(m, poly, 4×CH₃—CH₂—O_(poly(ethyl MTC))), 3.72-3.52 (m, poly,4×OCH₂CH_(2 PEG)+end groups), 3.38 (s, poly, 4×OCH_(3 poly(PEG MTC))),1.28-1.18 (m, poly, 2×CH_(3 poly(ethyl MTC))+end groups).

Example 11 ROP Polymerization of a Mixture of MTCOMPEG (M_(n)=0.5 k) andMTCOEt (Molar Ratio x:y=1:4, Respectively) Initiated byD4Bn4-[P(MTCOEt)_(n/4)]₄, Target DP=40

A portion of Example 8 in its solution containing both DBU and TU wasadded to a new flask. The portion was equivalent toD4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.070 g, 0.01mmol, 1 eq.). More DBU was added to obtain 0.1 eq. of DBU compared tothe monomers. MTCOMPEG (M_(n) 659 g/mol, 0.148 g, 0.22 mmol, 40 eq.) wasmixed with MTCOEt (MW 188 g/mol, 0.124 g, 0.66 mmol, 48 eq.) indichloromethane (2 mL). The solution with the monomers was mixed withinitiator D4Bn-[P(MTCOEt)_(n/4)]₄. After 3 hours the reaction wasquenched with benzoic acid and stirred for 30 min. The productD4Bn4-[P(MTCOEt)_(n/4)-b-P(MTCOMPEG_(x/4)-r-MTCOEt_(y/4))]₄ wasprecipitated in ethyl ether and dried in vacuum until a constant weightwas achieved. PDI=1.20. The structure is as shown in Example 9,differing by the x:y molar ratio of 1:4, and DP=40.

Example 12 ROP Polymerization of a Mixture of MTCOMPEG (M_(n)=0.5 k) andMTCOEt (Molar Ratio x:y=1:3, Respectively) Initiated byD4Bn-[P(MTCOEt)_(n/4)]₄, target DP=40

A portion of Example 8 in its solution containing both DBU and TU wasadded to a new flask. The portion was equivalent toD4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.080 g, 0.01mmol, 1 eq.). More DBU was added to obtain 0.1 eq. of DBU compared tothe monomers. MTCOMPEG (M_(n) 659 g/mol, 0.134 g, 0.20 mmol, 32 eq.) wasmixed with MTCOEt (MW 188 g/mol, 0.157 g, 0.84 mmol, 128 eq.) indichloromethane (2 mL). The solution with the monomers was mixed withinitiator D4Bn-[P(MTCOEt)_(n/4)]₄. After 3 hours the reaction wasquenched with benzoic acid and stirred for 30 minutes. The productD4Bn4P(MTCOEt)_(n/4)-b-P(MTCOMPEG_(x/4)-r-MTCOEt)₄ was precipitated inethyl ether and dried in vacuum until a constant weight was achieved.PDI=1.20. The structure is as shown in Example 9, differing by the x:ymolar ratio of 1:3, and DP=40. ¹H NMR (400 MHz, CDCl₃): delta=7.32, (b,20H, 4×Ar), 5.14 (s, 8H, 4×Ar—CH₂—O), 4.28 (m, poly,4×C—CH₂—O_(polymer backbone)), 4.18 (m, poly,4×CH₃—CH₂—O_(poly(ethyl MTC))), 3.72-3.52 (m, poly, 4×OCH₂CH_(2 PEG)+endgroups), 3.38 (s, poly, OCH_(3 poly(PEG MTC))), 1.28-1.21 (m, poly,2×CH_(3 poly(ethyl MTC))+end groups).

Example 13 ROP Polymerization of a Mixture of MTCOMPEG (M_(n)=2 k) andMTCOEt (Molar Ratio x:y=1:4, Respectively) Initiated byD4Bn-[P(MTCOEt)_(n/4)]₄, target DP=20

A portion of Example 8 in its solution containing both DBU and TU wasadded to a new flask. The portion was equivalent toD4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.200 g, 0.02mmol, 1 eq.). More DBU was added to have 0.05 eq. of DBU compared to themonomers. MTCOMPEG (M_(n) 2000 g/mol, 0.551 g, 0.28 mmol, 16 eq.) wasmixed with MTCOEt (MW 18⁸ g/mol, 0.189 g, 1.01 mmol, 64 eq.) indichloromethane (2 mL). The solution with the monomers was mixed withinitiator D4Bn-[P(MTCOEt)_(n/4)]₄. After 2 hours the reaction wasquenched with benzoic acid and stirred for 30 min. The productD4Bn4-[P(MTCOEt)_(n/4)-b-P(MTCOMPEG_(x/4)-r-MTCOEt_(y/4))]₄ wasprecipitated in ethyl ether and dried in vacuum until a constant weightwas achieved. PDI=1.27. The structure is as shown in Example 9,differing by the x:y molar ratio of 1:4, DP=20, and the M_(n)=2000 ofthe MPEG fragment. PDI=1.07. ¹H NMR (400 MHz, CDCl₃): delta=7.32, (b,20H, 4×Ar), 5.14 (s, 8H, 4×Ar—CH₂—O), 4.28 (m, poly,4×C—CH₂—O_(polymer backbone)), 4.18 (m, poly,4×CH₃—CH₂—O_(poly(ethyl MTC))), 3.84-3.44 (m, poly, 4×OCH₂CH_(2 PEG)+endgroups), 3.38 (s, 12H, 4×OCH₃), 1.28-1.18 (m, poly,2×CH_(3 poly(ethyl MTC))+end groups).

Example 14 ROP Polymerization of a Mixture of MTCOMPEG (M_(n)=5 k) andMTCOEt (Molar Ratio x:y=1:4, Respectively) Initiated byD4Bn-[P(MTCOEt)_(n/4)]₄, Target DP=20

A portion of Example 8 in its solution containing both DBU and TU wasadded to a new flask. The portion was equivalent toD4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.047 g, 0.004mmol, 1 eq.). More DBU was added to get 0.05 eq. of DBU compared to themonomers. MTCOMPEG (M_(n) 5000 g/mol, 0.305 g, 0.06 mmol, 16 eq.) wasmixed with MTCOEt (MW 188 g/mol, 0.045 g, 0.24 mmol, 64 eq.) in DCM (2mL). The solution with the monomers was mixed with initiatorD4Bn-[P(MTCOEt)_(n/4)]₄. After 2 hours the reaction was quenched withbenzoic acid and stirred for 30 min. The productD4-[Bn4P(MTCOEt)_(n/4)-b-P(MTCOMPEG_(x/4)-r-MTCOEt)_(y/4)]₄ wasprecipitated in ethyl ether and dried in vacuum until a constant weightwas achieved. The structure is as shown in Example 9, differing by thex:y molar ratio of 1:4, DP=20, and M_(n)=5000 of the MPEG fragment.

Example 15 Reaction of MTCOMPEG (M_(n)=5 k) withD4Bn-[P(MTCOEt)_(n/4)]₄, DP=1

That is, the tetrol D4Bn-[P(MTCOEt)_(n/4)]₄ was endcapped with a unit ofMTCOMPEG.

A portion of Example 8 in its solution containing both DBU and TU wasadded to a new flask. The portion was equivalent toD4Bn-[P(MTCOEt)_(n/4)]₄ (M_(n) 12597 g/mol, PDI 1.48, 0.100 g, 0.01mmol, 1 eq.). MTCOMPEG (M_(n) 5000 g/mol, 0.240 g, 0.05 mmol, 6 eq.) wasdissolved in dichloromethane (2 mL). The solution with MTCOMPEG wasmixed with initiator D4Bn-[P(MTCOEt)_(n/4)]₄. After 2 hours the reactionwas quenched with benzoic acid and stirred for 30 min. The productD4Bn-[P(MTCOEt)_(n/4)-b-P(MTCOMPEG)]₄ was precipitated in ethyl etherand dried in vacuum until a constant weight was achieved. PDI=1.12. ¹HNMR (400 MHz, CDCl₃): delta=7.32, (b, 20H, 4×Ar), 5.14 (s, 8H,4×Ar—CH₂—O), 4.28 (m, poly, 4×C—CH₂—O_(polymer backbone)), 4.18 (m,poly, 4×CH₃—CH₂—O_(poly(ethyl MTC))), 3.84-3.44 (m, poly,4×OCH₂CH_(2 PEG)+end groups), 3.38 (s, 12H, 4×OCH₃), 1.28-1.18 (m, poly,2×CH_(3 poly(ethyl MTC))+end groups).

Example 16 D4PEU Initiating the ROP of MTCOEt

D4PEU (MW 1604 g/mol, 0.090 g, 0.01 mmol, 1 eq.) was dissolved in dryTHF (1.786 mL, 1.9 M). (−)-sparteine (MW 234 g/mol, 0.042 g, 0.18 mmol,3 eq.) was added and stirred for 30 minutes. TU (MW 370 g/mol, 0.063 g,0.17 mmol, 3 eq.) and MTCOEt (MW 188 g/mol, 0.629 g, 3.34 mmol, 60 eq.)was added. After 5 hours the reaction was quenched with benzoic acid (MW102 g/mol, 0.180 g, 1.8 mmol, 30 eq.). Stirred for 30 min beforeprecipitation in 2-propanol. The product D4PEU-[P(MTCOEt)_(n/4)]₄ wasdried in vacuum until a constant weight was achieved.

Part 3. Large Stars. Divergent Approach.

Example 17 Ring Opening of the Cyclic Carbonate MTCOBn Initiated by DAB4to Form D64Bn

In a round bottom flask equipped with a stir bar was combined DAB64, ageneration 5 polypropyleneimine based dendrimer (0.15 g) having 64terminal amine groups and MTCOBn (0.347 g) in 3 mls of THF. The mixturewas stirred overnight to give D64Bn. The reaction was heated to 50° C.for 1 hour and the reaction mixture was added to a dialysis bag andtreated with methanol for 48 hours. The reaction was concentrated,redissolved in methylene chloride and stirred with CaH₂ for 24 hours,concentrated and stored in a glove box. ¹H NMR (400 MHz, DMSO):delta=7.38 (b, 320 H, 4×Ar), 7.05 (b, 4H, 64×OOC—NH), 5.05 (s, 120H,4×Ar—CH₂—OCO), 4.10+4.01 (d+d, 120H, 4×CH₂—OCO), 3.70 (m, 120H,4×CH₂—OH) 3.20 (m, H, 4×CH₂—NH—COO), 2.40 (m, H, 6×CH₂—N), 1.70-1.60 (m,12H, 6×CH₂), 1.20 (s, 64H, 4×CH₃).

Examples 18 Ring Opening Polymerization of L-Lactide Initiated by D64Bnto Form D64Bn-[P(LLA)_(n/64)]₆₄

Hydroxy functional dendrimer D64Bn (10 mg) prepared in Example 17 and(−)-sparteine (0.1 mg) were dissolved in methylene chloride and stirred.In another flask TU catalyst (0.1 mg) and L-lactide (LLA) (12.0 mg) weredissolved in CH₂Cl₂ (1 mL) and added to the initiator flask. After 2.5hours benzoic acid (10 mg) was added and the polymerD64Bn-[P(LLA)_(n/64)]₆₄ was precipitated in methanol. Mn=118000,PDI=1.3.

Example 19 Ring Opening of the Cyclic Carbonate MTCODNT Initiated byDAB64 to form D64DNT

To a round bottom flask equipped with a stir bar, DAB64 (0.05 g) wascombined together with MTCODNT (0.173 g) in THF and stirred overnight(50° C.) to give D64DNT. The reaction mixture was added to a dialysisbag and treated with methanol for 48 hours. The reaction wasconcentrated, redissolved in THF and stirred with CaH₂ for 24 hours,concentrated and stored in a glove box. ¹H NMR (400 MHz, DMSO): δ=8.8,(64 H, Ph), 8.4 (64 H, Ph), 7.95 (64 H, Ph), 6.95 (64 H, NH), 4.9 (128H,CH₂ OCO), 4.30 (128H CH2ChO), 3.95 (128H CH2OH), 2.9, 2.2 and 1.5(dendrimer core) 1.0 (192H CH3).

Example 20 Ring Opening Polymerization of L-Lactide Initiated by D64DNTto Form D64DNT-[P(LLA)_(n/64)]₆₄

The procedure described in Example 18 was repeated using the hydroxyfunctional dendrimer D64DNT produced in Example 19 to formD64DNT-[P(LLA)_(n/64)]₆₄. Mn=122000, PDI=1.2.

Example 21 Ring Opening of the Cyclic Carbonate MTCU Initiated by DAB64to Form D64PEU

To a round bottom flask equipped with a stir bar, DAB64 (0.15 g) wascombined together with MTCU (0.1449 g) and stirred overnight (50° C.) togive D64PEU. The reaction mixture partially fell out of solutionovernight and the slurry was precipitated in methylene chloride, driedand stored in the box. ¹H NMR (400 MHz, DMSO): 8.6 (m, 64H NH), 7.4(128H, Ph), 7.18 (128H, Ph), 7.05 (64H, CONH), 6.95 (64H, Ph), 6.2 (64H,CONHCH2), 5.05 and 4.95 (128H, CH2OCOH), 4.0-4.3 (256H, CH2OH, CH2OC),2.95, 2.20 and 1.50 (dendrimer core).

Example 22 Ring Opening Polymerization of L-Lactide Initiated by D64PEUto Form D64PEU-[P(LLA)_(n/64)]₆₄

The procedure described in Example 18 was repeated using the hydroxyfunctional dendrimer D64PEU produced in Example 21 to formD64PEU-[P(LLA)_(n/64)]₆₄. Mn=160000, PDI=1.18.

Part 4. Antimicrobial Star-Shaped Polymers

Example 23 Ring opening polymerization of L-lactide initiated by D4Bn toform D4Bn-[P(LLA)_(n/4)]₄

D4Bn (32.6 mg, 0.02 mmol), L-lactide (LLA) (145 mg, 1.0 mmol), TU (10.6mg, 0.03 mmol) were dissolved in dry methylene chloride (1.0 mL) andtransferred to a vial containing (−)-sparteine (2.9 mg, 0.01 mmol) toconduct ROP at room temperature for 2 hours in the globe box([LLA]/[I]=40). Benzoic acid (11 mg, 0.09 mmol) was added to quench thereaction (˜99% conversion). The mixture was stirred for 30 min,precipitated in 2-propanol, isolated, and dried in vacuum for 20 hoursto yield D4Bn-[P(LLA)_(n/4)]₄ (97 mg, 55%). GPC (THF, PS standard):M_(n)=7900, PDI=1.19. ¹H NMR (400 MHz, CDCl₃): delta 7.39-7.27 (m, 20H;Ph), 5.25-5.09 (m, ˜80H; PhCH₂ and CH_(PLA)), 4.42-4.10 (m, 20H; OCH₂and CH_(end group)), 3.23-3.06 (b, 6H; CH₂NH), 2.51-2.27 (b, 12H; NCH₂),1.63-1.33 (m, ˜263H; CH₂ and CH_(3 P(LLA))), 1.29-1.18 (m, 12H; CH₃).

Example 24 Ring Opening Polymerization of the Cyclic Carbonate MTCOPrBrInitiated by D4Bn-[P(LLA)_(n/4)]₄ to formD4Bn-[P(LLA)_(n/4)-b-P(MTCOPrBr)_(m/4)]₄

D4Bn-[P(LLA)_(n/4)]₄ (65 mg, [OH]=0.04 mmol), MTCOPrBr (126 mg, 0.45mmol), and TU (4.1 mg, 0.01 mmol) were dissolved in dry methylenechloride (1.5 mL) and transferred to a vial containing DBU (1.9 mg, 0.01mmol) to conduct ring opening polymerization at room temperature for 3hours in the globe box ([MTCOPrBr]/[OH]=11). Acetic anhydride (13 mg,0.13 mmol) was added to the mixture in order both to quench the reactionand to cap the hydroxyl groups (˜84% conversion). The mixture wasstirred for 40 hours, precipitated in 2-propanol, isolated, and dried invacuum for 50 hours to yield D4Bn-[P(LLA)_(n/4)-b-P(MTCOPrBr)_(m/4)]₄(146 mg, 77%). GPC (THF, PS standard): M_(n)=15200, PDI=1.32. ¹H NMR(400 MHz, DMSO-d₆): delta 7.38-7.28 (m, 20H; Ph), 5.24-5.08 (m, ˜80H;PhCH₂ and CH_(P(LLA))), 4.45-4.11 (m, ˜293H; OCH₂ andOCH_(2 P(MTCOPrBr))), 3.51-3.39 (m, ˜91H; CH₂Br_(P(MTCOPrBr))),3.21-3.08 (b, 5H; CH₂NH), 2.48-2.32 (b, 11H; NCH₂), 2.24-2.14 (m, ˜96H;CH_(2 P(MTCOPrBr))), 1.65-1.45 (m, ˜251H; CH₂ and CH_(3 P(LLA))), 1.39(b, 4H; CH₂), 1.30-1.18 (m, ˜155H; CH₃ and CH_(3 P(MTCOPrBr))).

Example 25 Quaternization of D4Bn4-[P(LLA)_(n/4)-b-P(MTCOPrBr)_(m/4)]₄

Trimethylamine gas (301 mg, 5.1 mmol) was charged to an acetonitrilesolution (6 mL) of D4Bn-[P(LLA)_(n/4)-b-P(MTCOPrBr)_(m/4)]₄ (119 mg,[Br]=0.29 mmol) immersed in a dry-ice/acetone bath. The solution wasallowed to warm to room temperature and with continued stirring for 16hours. Then acetonitrile and excess gasses were removed from thereaction mixture under vacuum to giveD4Bn-[P(LLA)_(n/4)-b-P(MTCOPrBr*NMe₃)_(n/4)] (134 mg, 99%). M_(n) 21800(calculated from NMR). ¹H NMR (400 MHz, MeOH-d₄): delta 7.43-7.27 (b,20H; Ph), 5.30-5.12 (m, ˜87H; PhCH₂ and CH_(P(LLA))), 4.51-4.14 (m,˜356H; OCH₂ and OCH_(2 P(MTCOPrBr*NMe3))), 3.64-3.44 (b, ˜112H;CH₂Br_(P(MTCOPrBr*NMe3))), 3.29-3.11 (b, ˜499H; CH₂NH andN⁺CH_(3 P(MTCOPrBr*NMe3))), 2.64-2.41 (b, 7H; NCH₂), 2.33-2.15 (b,˜110H; CH_(2 P(MTCOPrBr*NMe3))), 1.63-1.37 (m, ˜291H; CH₂ andCH_(3 P(LLA))), 1.39-1.18 (m, ˜192H; CH₃ and CH_(3 P(MTCOPrBr*NMe3))).

Surface Modified Inorganic Nanoparticles

Example 26 Synthesis of CoFe₂O₄ Nanoparticles

In a flask, 2 mmol Fe(acac)₃, 1 mmol Co(acac)₂, 10 mmol1,2-hexadecanediol, 6 mmol oleic acid, 6 mmol oleylamine, and 20 mL ofbenzyl ether were combined and mechanically stirred under a flow of N₂.The mixture was heated to 200° C. for 2 hours and then, under a blanketof N₂, heated to reflux (˜300° C.) for 1 hour. The black colored mixturewas cooled to room temperature by removing the heat sources. Underambient conditions, 40 mL of ethanol was added to the mixture and ablack material was precipitated and separated via centrifugation at 6000rpm for 10 minutes. The black precipitate was dissolved in hexane with0.1% oleic acid. The mixture was centrifuged at 6000 rpm for 10 minutesto remove any undispersed residue. The product was then precipitatedwith ethanol, centrifuged to remove the solvent, and dried in vacuumovernight. The average diameter of the CoFe₂O₄ nanoparticles is 6 nmwith narrow size distribution. The as-synthesized 6 nm CoFe₂O₄nanoparticles were further used as the seeds to grow larger particles.Typically, 2 mmol Fe(acac)₃, 1 mmol Co(acac)₂, 10 mmol1,2-hexadecanediol, 2 mmol oleic acid, 2 mmol oleylamine, and 20 mL ofbenzyl ether were mixed and mechanically stirred under a flow of N₂. 6mL of the above synthesized 6 nm CoFe₂O₄ nanoparticle hexane solution(15 mg/mL) was added. The mixture was first heated to 100° C. for 30 minto remove hexane, and then increased to 200° C. for 1 hour. Under ablanket of N₂, the mixture was further heated to 300° C. for 30 min.Following the same workup procedures, the monodispersed CoFe₂O₄nanoparticles with a diameter of 15 nm were obtained. Finally, this seedmediated growth method was repeated again to prepare 18 nm monodispersedCoFe₂O₄ nanoparticles.

Example 27 Silica Shell-Coated CoFe₂O₄ Nanoparticle with AmineFunctionalized Surface

In a glass container under ambient conditions, 1 mL of polyacrylic acid(PAA) in THF (10 mg/mL) was added to a dispersion of above synthesized18 nm CoFe₂O₄ Nanoparticles (10 mg in 10 mL). The mixture was shaken for2 hours with occasional sonication. The modified particles wereseparated with a magnet and the solvent was decanted. The particles werewashed three times with hexane and methanol to remove the free oleicacid and excess PAA polymer. The washed particles were dispersed inaqueous solution by ionizing the carboxylic groups with a dilute NaOHsolution. Then, 1.5 mL of PAA modified CoFe₂O₄ nanoparticle aqueoussolution was mixed with ethanol (10 mL), and ammonium hydroxide (30 wt%, 400 microliters) by vigorous mechanical stirring. Tetraethoxy silane(TEOS)/ethanol solution (200 microliters, 10 mM) was added to themixture every 2 hours until the total amount of TEOS solution reached 1mL. After obtaining the desired size, aminopropyl triethoxysilane wasadded to the solution. The amine-functionalized silica coated CoFe₂O₄nanoparticles were collected by magnetic separation, and washed withethanol three times, and finally dispersed in THF.

Example 28 Ring Opening Reaction of MTCOMPEG with Amine-FunctionalizedSilica Coated CoFe₂O₄ Nanoparticles (Example 27)

MTCOMPEG (M_(n) 2000 g/mol, n˜44, PDI 1.04, 50 mg, 0.025 mmol) was addedto a suspension of the amine-functionalized silica coated CoFe₂O₄nanoparticles in THF. The reaction was heated to dissolve the MTCOMPEGthen stirred for 16 hours at ambient temperature. The functionalizednanoparticles were collected by centrifugation.

Example 29 Ring Opening Polymerization of L-Lactide from MTCOMPEGFunctionalized CoFe₂O₄ Nanoparticles (Example 28)

A solution of MTCOMPEG-functionalized nanoparticles from Example 28 indichloromethane (1 mL) was transferred into the glove box. L-Lactide(12.7 mg), 10 microliters of a thiourea catalyst solution (15.4 mg/mL indichloromethane), and 10 microliters of a DBU solution (5.9 mg/mL indichloromethane) were added to the solution of MTCOMPEG-functionalizednanoparticles. The reaction mixture was stirred at ambient temperaturefor 16 hours, then quenched with benzoic acid (MW 102 g/mol, 0.021 g,0.21 mmol, 20 eq.). The product CoFe₂O₄-MPEG-[P(LLA)] was isolated bycentrifugation.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiments were chosen and described in order to best explain theprinciples of the invention and their practical application, and toenable others of ordinary skill in the art to understand the invention.

What is claimed is:
 1. A method, comprising: independently covalentlyattaching by a ring opening reaction a first cyclic carbonyl monomer tothree or more independent nucleophilic surface groups of a nanostructureselected from the group consisting of i) macromolecules selected fromthe group consisting of crosslinked polymers, latexes, hyperbranchedpolymers, dendrimers, dendrons, cyclodextrins, and combinations of theforegoing materials and ii) inorganic materials, thereby forming a firstmodified nanoparticle, the first modified nanoparticle comprising threeor more initiator groups produced by the ring opening of the firstcyclic carbonyl monomer; and initiating a ring opening polymerization ofone or more cyclic carbonyl monomers by the three or more initiatorgroups, thereby forming a surface modified nanoparticle comprising threeor more independent surface branches, wherein each of the three or moreindependent surface branches comprises a first polymer produced by thering opening polymerization; wherein the first cyclic carbonyl monomerhas formula (12):

wherein each Y is independently selected from the group consisting of—O—, —NH—,

 and —S—, R⁵ and R⁶ are independent monovalent radicals selected fromthe group consisting of hydrogen, and monovalent hydrocarbon radicalscomprising 1 to 30 carbons, and M¹ is a monovalent radical selected fromthe group consisting of —OR¹, —NHR¹, —NR¹R¹, and —SR¹, wherein R¹ is amonovalent radical.
 2. The method of claim 1, wherein each Y of formula(12) is —O—.
 3. The method of claim 1, wherein one Y of formula (12) is—O— and another Y is —NH—.
 4. The method of claim 1, wherein one Y offormula (12) is —O— and another Y is —S—.
 5. The method of claim 1,wherein each Y of formula (12) is —O—, and R⁵ is methyl, ethyl, orpropyl.
 6. The method of claim 1, wherein each Y of formula (12) is —O—,and R⁵ is hydrogen.
 7. The method of claim 1, wherein the first cycliccarbonyl monomer is a cyclic carbonate, and M¹ comprises a secondpolymer.
 8. The method of claim 7, wherein the second polymer is apoly(alkylene ether).
 9. The method of claim 1, wherein the first cycliccarbonyl monomer does not comprise a polymeric substituent.
 10. Themethod of claim 1, wherein one or more of the one or more cycliccarbonyl monomers comprises a functional group comprising apoly(alkylene ether).
 11. The method of claim 1, wherein each of thethree or more independent surface branches comprises a repeat unitcomprising a side chain comprising a monovalent leaving group, and themethod further comprises treating the surface modified nanoparticle witha tertiary amine, thereby forming a second surface modified nanoparticlecomprising a quaternary amine.
 12. The method of claim 1, wherein thefirst polymer is a block copolymer.
 13. The method of claim 1, whereinthe nanostructure is a dendrimer selected from the group consisting ofDAB4, DAB8, DAB16, DAB32, DAB64, and combinations thereof.
 14. Themethod of claim 1, wherein the nanostructure comprises an inorganicmaterial selected from the group consisting of cobalt, iron, andcombinations thereof.
 15. The method of claim 1, wherein the firstcyclic monomer is a member of the group consisting of

and combinations thereof.
 16. The method of claim 1, further comprisingforming a first aqueous mixture comprising the surface modifiednanoparticle, and contacting the first aqueous mixture with a secondaqueous mixture comprising a biologically active material, therebyforming a loaded nanoparticle.
 17. The method of claim 16, wherein thebiologically active material is a gene.
 18. The method of claim 16,wherein the biologically active material is a drug.
 19. The method ofclaim 16, further comprising contacting a cell with the loadednanoparticle.